FOOD QUALITY

Edited by Kostas Kapiris

FOOD QUALITY
Edited by Kostas Kapiris
Food Quality
Edited by Kostas Kapiris

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First published April, 2012

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Food Quality, Edited by Kostas Kapiris

p. cm.
ISBN 978-953-51-0560-2
Contents

Preface IX

Section 1 Molecular Approaches to Achieve the Food Quality 1

Chapter 1 Strategies for Iron Biofortification of Crop Plants 3

Mara Schuler and Petra Bauer

Chapter 2 Monitoring Harmful Microalgae

by Using a Molecular Biological Technique 15
Tomotaka Shiraishi, Ryoma Kamikawa,
Yoshihiko Sako and Ichiro Imai

Chapter 3 Species Identification

of Food Spoilage and Pathogenic Bacteria
by MALDI-TOF Mass Fingerprinting 29
Karola Böhme, Inmaculada C. Fernández-No,
Jorge Barros-Velázquez, Jose M. Gallardo,
Benito Cañas and Pilar Calo-Mata

Chapter 4 Raman Spectroscopy: A Non-Destructive

and On-Site Tool for Control of Food Quality? 47
S. Hassing, K.D. Jernshøj and L.S. Christensen

Chapter 5 Contamination of Foods by Migration

of Some Elements from Plastics Packaging 73
O. Al-Dayel, O. Al-Horayess, J. Hefni,
A. Al-Durahim and T. Alajyan

Section 2 Some Case Studies Improving the Food Quality 81

Chapter 6 Senescence of the Lentinula edodes

Fruiting Body After Harvesting 83
Yuichi Sakamoto, Keiko Nakade,
Naotake Konno and Toshitsugu Sato
VI Contents

Chapter 7 Feeding Habits of Both Deep-Water

Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae)
in the Ionian Sea (E. Mediterranean) 111
Kostas Kapiris
Preface

Food quality is the quality characteristics of food that is acceptable to consumers. This
includes external factors as appearance (size, shape, colour, gloss, and consistency),
texture, and flavour; factors such as federal grade standards (e.g. of eggs) and internal
(chemical, physical, microbial).

Food quality is an important food manufacturing requirement, because food

consumers are susceptible to any form of contamination that may occur during the
manufacturing process. Many consumers also rely on manufacturing and processing
standards, particularly to know what ingredients are present, due to dietary,
nutritional requirements, or medical conditions (e.g., diabetes, or allergies). Food
quality also deals with product traceability, e.g. of ingredient and packaging suppliers,
should a recall of the product be required. It also deals with labeling issues to ensure
there is correct ingredient and nutritional information.

Besides ingredient quality, there are also sanitation requirements. It is important to

ensure that the food processing environment is as clean as possible in order to produce
the safest possible food for the consumer. Foodborne diseases due to microbial
pathogens, biotoxins, and chemical contaminants in food represent serious threats to
the health of thousands of millions of people. Serious outbreaks of foodborne disease
have been documented on every continent in the past decades, illustrating both the
public health and social significance of these diseases. A recent example of poor
sanitation has been the 2006 North American E. coli outbreak involving spinach, an
outbreak that is still under investigation after new information has come to light
regarding the involvement of Cambodian nationals. Foodborne diseases not only
significantly affect people's health and well-being, but they also have economic
consequences for individuals, families, communities, businesses and countries. These
diseases impose a substantial burden on healthcare systems and markedly reduce
economic productivity. Poor people tend to live from day to day, and loss of income
due to foodborne illness perpetuates the cycle of poverty.

Effective national food control systems are essential to protect the health and safety of
domestic consumers. Governments all over the world are intensifying efforts to
improve food safety in response to an increasing number of problems and growing
consumer concerns in regards to various food risks. Responsibility for food control in
X Preface

most countries is shared between different agencies or ministries. The roles and
responsibilities of these agencies may be quite different, and duplication of regulatory
activity, fragmented surveillance and a lack of coordination are common.

The Food and Agriculture Organization of the United Nations (FAO) and the World
Health Organization (WHO) have a strong interest in promoting national food control
systems that are based upon scientific principles and guidelines, and which address all
sectors of the food chain. This is particularly important for developing countries as
they seek to achieve improved food safety, quality and nutrition, but will require a
high level of political and policy commitment.

During the recent past new analytical approaches used to assess the quality of foods
have been emerging, new molecules have been discovered, and there have been many
advances in molecular biology and genetics. As well as comparing and evaluating
indices used to assess quality of foods, this book offers some recently developed
techniques and methods. The book discusses the potential of these novel approaches,
which attempt to solve the existent problems and offer to the food scientist valuable
assistance for the future. The detailed methodologies and their practical applications
could consist a fundamental reference work for industry and a requisite guide for the
research worker, food scientist and food analyst. It will serve as a valuable tool for the
analysts improving their knowledge with new scientific data for quality evaluation.
Except the above laboratory techniques’ descriptions, two case studies chapters
provide data on the improvement of food quality in the natural environment: the
study of the postharvest spoilage, such as browning of the gills and softening of the
fruiting body of land (mushrooms) and the improved food quality of the preys of
marine (deep water shrimps) organisms.

The World Food Summit of 1996 defined food security as existing “when all people at
all times have access to sufficient, safe, nutritious food to maintain a healthy and active
life”. Commonly, the concept of food security is defined as including both physical
and economic access to food that meets people's dietary needs as well as their food
preferences.

Dr. Kapiris Kostas

Hellenic Center for Marine Research (H.C.M.R.),
Institute of Marine Biological Resources (I.M.B.R.),
Athens,
Greece
 Section 1

Molecular Approaches
to Achieve the Food Quality
 1

Strategies for Iron

Biofortification of Crop Plants
Mara Schuler and Petra Bauer
Dept. Biosciences-Plant Biology,
Saarland University,
Saarbrücken,
Germany

1. Introduction
Iron (Fe) is an essential element for all living organisms because of its property of being able
to catalyze oxidation/reduction reactions. Fe serves as a prosthetic group in proteins to
which it is associated either directly or through a heme or an iron-sulfur cluster. It exists in
two redox states, the reduced ferrous Fe2+ and the oxidized Fe3+ form and is able to loose or
gain an electron, respectively, within metalloproteins (e.g. Fe-S cluster or heme-Fe proteins).
Such metalloproteins are involved in fundamental biochemical reactions like the electron
transfer chains of respiration and photosynthesis, the biosynthesis of DNA, lipids and other
metabolites, the detoxification of reactive oxygen species.
The cellular processes that involve Fe take place in distinct intracellular compartments like
e.g. cytoplasm, mitochondria, plastids, cell walls, which therefore need to be provided with
an adequate amount of Fe. Since this metal is involved in a wide range of essential
processes, the undersupply with Fe leads to severe deficiency symptoms in the affected
organism.
Fe deficiency is one of the most prevalent and most serious nutrient deficiencies threatening
human health in the world, affecting approximately two billion people (de Benoist et al.,
2008). Various physiological diseases, such as anaemia and some neurodegenerative
diseases are triggered by Fe deficiency (Sheftela et al., 2011). Especially those countries are
affected by Fe deficiency diseases, where people have low meat intake and the diets are
mostly based on staple crops. Young children, pregnant and postpartum women are the
most commonly and severely affected population groups, because of the high Fe demands
of infant growth and pregnancy (de Benoist et al., 2008). Human health problems caused by
Fe deficiency can be prevented by specific attention to food composition and by choosing a
balanced diet with sufficient and bio-available Fe content.
Several possibilities exist to enrich the diet with bio-available Fe, which all have advantages
and disadvantages. Supplementation of Fe in the diet is possible by supply of Fe chelates
and salts in form of pills (Yakoob & Bhutta, 2011). However, formulations which are well
tolerated by patients are expensive and particularly in underdeveloped areas of the world
difficult to supply daily, as additional systems for purchasing, transport and distribution
4 Food Quality

have to be established, associated with extra costs. The fortification of food products like
flour with Fe salts is also effective (Best et al., 2011) and in place in some developed
countries (Huma et al., 2007). Generally, an existing food industry is required for food
processing, so that again supply is difficult in underdeveloped countries. The diversification
of the diet with an emphasis on improvement of Fe-rich food crops like certain green leafy
vegetables and legume seeds would be highly effective and desirable. In fact, it is actually
the simplification of the diet with its low diversification that is the main cause of the
micronutrient deficiency (Nair & Iyengar, 2009). The structure of agriculture, the green
revolution and the need to supply sufficient food in light of a rapidly increasing world
population had caused a concentration on calorie-rich carbohydrate-providing crops
(Gopalan, 1996). Finally, the bio-fortification of staple crops is considered to be a very
effective method which would reach many people even in underdeveloped countries (Bouis
et al., 2011). A prerequisite is that the local staple crops are bio-fortified so that they contain
more and better available Fe. This can generally be reached by breeding, which is performed
either by the breeding industry or by governmental agencies. The newly bred lines need to
be distributed to and accepted by the local farmers. In any case, it seems that the prevention
of Fe deficiency in the population of underdeveloped countries may strongly depend on
governmental willingness, administration and regulation concerning the quality and
quantity of food. It is clear that none of the above mentioned treatments is “cheap”. Yet, the
economic losses due to fatigue and neuronal dysfunctions might be far greater in negative
value than the expected expenses to prevent these problems (Hunt, 2002). Therefore, the
combat against Fe deficiency diseases is among the top priorities particularly listed by the
WHO (de Benoist et al., 2008).
Here, we present some of the approaches for bio-fortification of crops with Fe. This report
will focus on the underlying technological advances and our knowledge about the
physiological processes leading to the enrichment of specific plant organs with Fe and their
increased bio-availability.

2. Overview about Fe homeostasis in plants

The most important plants for nutrition of humans and mammals are the highly evolved
flowering plants (angiosperms). These include the major crops and plant model organisms
like rice, maize, legumes and Arabidopsis thaliana. Fe is found in all plant organs, which
include roots, leaves, flowers, fruits with seeds, storage organs like tubers. Depending on
the plant crop species and its use all these various parts can be edible, and in this case the
concentrations of bio-available Fe should be high. Under natural conditions, all Fe of living
organisms ultimately enters the nutrition chain via plant roots. In the soil, Fe mainly exists
as Fe3+, often bound as iron hydroxides in mineral soil particles (Marschner, 1995). Plants
need a Fe concentration of 10-6 M for optimal growth, but the concentration of free Fe3+ in an
aerobic, aqueous environment of the soil with a pH of 7 is about 10-17 M. At lower pH the
solubility of Fe is increased, but a Fe3+ concentration of 10-6 M is reached at pH 3,3 (Hell &
Stephan, 2003). 30% of the world`s crop land is too alkaline for optimal plant growth.
Moreover, it appears that some staple crops, like rice, are especially susceptible to Fe
deficiency. Under alkaline and calcareous soil conditions, bioavailable Fe concentrations are
low in the soil despite of the abundance of this metal in the earth crust. To meet their
demand for Fe, plants need to mobilize Fe in the soil by rendering it more soluble before
Strategies for Iron Biofortification of Crop Plants 5

they are able to take it up into their roots. Two effective Fe acquisition systems known as
Strategy I and Strategy II have evolved in higher plants, based on reduction and chelation of
Fe3+, respectively (Römheld, 1987; Römheld & Marschner, 1986). The group of strategy I
plants includes all dicotyledonous and all non-grass monocotyledonous plants. They acidify
the soil, reduce Fe3+ and take up divalent Fe2+ via specific divalent metal transporters (Jeong
& Guerinot, 2009; Morrissey & Guerinot, 2009). All monocotyledonous grasses are Strategy
II plants, including all major cereal crop plants like rice (Oryza sativa), barley (Hordum
vulgare), wheat (Triticum aestivum) and maize (Zea mays). These plants synthesize and secrete
Fe3+-chelating methionine derivatives termed phytosiderophores of the mugineic acid
family and subsequently take up Fe3+-phytosiderophore complexes (Jeong & Guerinot, 2009;
Kobayashi et al., 2010; Morrissey & Guerinot, 2009). Fe reaches leaves mainly in complexed
form with citrate through the xylem, which is a plant conductive tissue for water and
mineral long-distance transport. Typical sink organs like immature organs receive Fe via the
phloem pathway, which represents the conductive tissue for assimilates and signals. Inside
plants, Fe is distributed to all tissues and cellular compartments through the activities of
several different types of membrane-bound metal transport proteins (Curie et al., 2009;
Jeong & Guerinot, 2009). Metal ions are predominant in a bound or chelated form inside
cells to enhance solubility and transport but at the same time minimize toxicity effects of
metal ions. In plants, oganic acids like citrate and malate, the amino acid histidine and the
plant-specific methionine derivative nicotianamine are mainly involved in Fe transport and
solubility (Briat et al., 2007; Callahan et al., 2006). Chelators for metals also include
polypeptides such as phytochelatins (PCs) and metallothioneins (MTs) which are essentially
involved in the tolerance to potentially toxic heavy metal ions (Hassinen et al., 2011; Pal &
Rai, 2010). Fe can be stored in form of ferritin in the plastids which also serves to reduce
oxidative stress (Briat et al., 2010b). In the vacuole Fe is frequently bound by phytic acid,
which is composed of inositol esterified with phosphorous acid. The ionized form binds
several mineral ions including Fe. It is present in cereal grains, nuts and leguminous seeds
(Gibson et al., 2010).
In conclusion, plants contain a complex regulation network of genes which provide uptake,
chelation, transport, sub-cellular distribution and the storage of Fe. Knowing these processes
is the prerequisite for their manipulation in order to breed in the future high-quality
nutritious crops.

3. Biofortification strategies
Bio-fortification designates the natural enrichment of plants with nutrients and health-
promoting factors during their growth. Bio-fortification focuses on generating and breeding
major staple food crops that would produce edible products enriched in bioavailable
amounts of micronutrients, provitamin A carotenoids or several other known components
that enhance nutrient use efficiency and are beneficial to human health (Hirschi, 2009).
The bio-fortification approach is interesting for staple crops that were mainly bred for
carbohydrate content, processing characteristics and yield in the past decades, e.g. maize,
wheat, rice and also some of the local plants like Cassava, potato and sweet potato. Elite
lines highly performing in the field might on the other hand be poor in micronutrient
contents (White & Broadley, 2009). Plants with a higher nutritional value can be produced
6 Food Quality

by classical breeding. In this case, wild relatives or varieties with beneficial micronutrient
content are selected and the respective trait crossed into the elite lines. This approach is
labor-intensive, it can be aided by the usage of molecular markers that are closely linked
with the traits of interest; in an optimal case, the molecular nature of the trait is known and
can be followed directly with molecular PCR and sequencing technologies in the various
breeding steps (Tester & Langridge, 2010; Welch & Graham, 2004). Alternatively, bio-
fortified crops with new properties can be generated using gene technology in addition to
classical breeding. In this case, the trait of interest is constructed in vitro using molecular
cloning to combine promoters and genes that together confer the trait. These constructs are
transferred into the crops, which could be achieved for example by biolistic methods based
on the bombardment of plant cells with the DNA or using as tool Agrobacterium tumefaciens.
The integration event of the DNA fragment conferring the new trait into the plant genome is
selected, respective transgenic plants are generated and multiplied (Sayre et al., 2011;
Shewry et al., 2008). Research on bio-fortification via classical breeding and/or gene
technology-based breeding was stimulated by non-profit funding organizations, such as
through the program HarvestPlus (http://www.harvestplus.org) (Bouis et al., 2011) and the
Golden rice project (http://www.goldenrice.org) (Beyer, 2010). Bio-fortification thus
became an agricultural and breeding tool to combat human malnutrition in the world.
For the Fe bio-fortification breeding, several challenges have to be overcome which can be
mastered if scientists acquire a better understanding of the physiological mechanisms of
plant metal homeostasis and political regulations allow for distributing such modified
plants (Hotz & McClafferty, 2007). First, the plants have to increase Fe uptake. Depending
on the soil properties, specific strategies for Fe mobilization in the soil have to be employed
by the plants. Plants are then able to render Fe in the soil more soluble and bio-available to
them. Second, Fe should accumulate in the edible parts of the plant such as seeds and fruits.
These plant parts should act as effective sinks for Fe. Third, the nutrients should be
preferentially stored in a form that renders them bioavailable for the human digestive
system. Fe can be complexed with soluble organic ligands which would increase its bio-
availability. However, some compounds like phytic acid can precipitate Fe and act as
antinutrients if phytase is not provided.
First attempts to target physiological processes of Fe homeostasis have already been started
to test the effect on bio-fortification. Moreover, assays are available to test for uptake of Fe
from plant food items (Glahn et al., 2002; Lee et al., 2009; Maurer et al., 2010).

4. Examples for Fe biofortification research in plants

4.1 Reduction of phytic acid content
A successful approach for Fe bio-fortification relies on the reduction of Fe complex-forming
metabolites that act as anti-nutrients, like tannins, a phenolic polymer, and phytic acid
(Welch & Graham, 2004). Phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate; InsP6)
comprises up to 80 % of the total seed phosphorus content and its dry mass may account for
1-2 % of seed weight (Hurrell, 2002). It accumulates as a phosphorous and mineral storage
compound in globoids in the seeds of many staple crops, including legumes like soybean,
cereal embryo and/or aleurone cells (Bohn et al., 2008). In developing countries, the
prevalence of phytic acid in the plant-based diet is believed to contribute to the high rate of
Strategies for Iron Biofortification of Crop Plants 7

Fe deficiency and anemia. On the other hand, reduction of phytic acid contents is also seen
negative, since in a well-balanced diet it has health-promoting effects on the immune system
and in preventing kidney stones (Shamsuddin, 2008). Phytic acid content can be reduced by
disruption of its biosynthetic chain which would result in a “low phytic acid” (lpa)
phenotype (Raboy, 2007; Rasmussen et al., 2010). Phytic acid is mainly synthesized from d-
glucose-6-phosphate transformed first into 1d-myo-inositol-3-phosphate [Ins(3)P1] (Loewus
& Murthy, 2000). Several biochemical pathways seem to be involved in transforming
Ins(3)P1 to InsP6 in plants, depending on the plant species (Bohn et al., 2008; Rasmussen et
al., 2010). Furthermore, an ABC transporter is required for transport and
compartmentalization in the final steps which can also be disrupted (Shi et al., 2007). Several
mutant lines have been identified in various plant species including soybean (Hitz et al.,
2002; Wilcox et al., 2000), maize (Pilu et al., 2003; Raboy et al., 2000), wheat (Guttieri et al.,
2004), rice (Larson et al., 2000; Liu et al., 2007) and Arabidopsis (Kim & Tai, 2011; Stevenson-
Paulik et al., 2005). However, conventional breeding may result in strong phytic acid
reduction and thereby in counteracting effects of such lpa mutants, like decreased
germination and reduced seedling growth, if the effect takes place overall in the plants.
Better mutants can be created using gene technology since only the late functions of the
genes for phytate synthesis may be abolished and only in certain phases and organs during
the life cycle of the plants by using specific promoters that allow expression of the
transgenes under very controlled conditions (Kuwano et al., 2009; Kuwano et al., 2006).
Alternatively, the late stages of phytic acid biosynthesis and transport may be specifically
targeted in mutants (Stevenson-Paulik et al., 2005). For example, two Arabidopsis genes for
inositol polyphosphate kinases, ATIPK1 and ATIPK2, have been disrupted, which are
required for the later steps of phytic acid synthesis. These mutants were found to produce
93 % less phytic acid in seeds, while seed yield and germination were not affected. It was
however found that the loss of phytic acid precursors altered phosphate sensing.
An alternative approach may rely on the transformation of plants with phytase enzymes.
Such enzymes are isolated from a multitude of different microorganisms, and heat-stability
besides enzyme activity are important criteria to consider in the food processing procedure
(Bohn et al., 2008; Rao et al., 2009).
Numerous examinations have to follow to find a solution to exclude negative influences of
phytic acid as an anti-nutrient but sustain its positive effects on plant growth. It has to be
investigated in future studies how useful phytate-reduced crops are for human Fe uptake.

4.2 Increase of ferritin content

Ferritins are multiprotein complexes consisting of ferritin peptide chains that are organized
in globular manner to contain inside up to 4000 Fe3+ ions. Existing reports suggest that Fe is
stored short- and long-term in ferritins and utilized for the accumulation of Fe-containing
proteins. This way, ferritins supply Fe during developmental processes of plants, and some
plant species contain high ferritin-Fe levels in seeds (Briat et al., 2010a). Ferritins also serve
to alleviate oxidative stress (Briat et al., 2010b). However, not in every case high ferritin
levels need to colocalize with high Fe levels in seeds (Cvitanich et al., 2010). Ferritin-Fe is
separated from other Fe-binding components by its protein coat and its localization inside
plastids or mitochondria. Ferritins exist in all organisms as a store of Fe. Ferritins in general
8 Food Quality

and ferritins in plant food items provide a high Fe bioavailability (Murray-Kolb et al., 2002;
San Martin et al., 2008; Theil, 2004).
Ferritin genes were used in bio-fortification approaches. For example, leguminous ferritin
genes, especially from soybean and bean, were over-expressed in plants, and subsequently
an accumulation of ferritin protein was observed in the plants. Ferritins from legumes had
been used since this plant family contains high ferritin levels in seeds, and the legume seeds
serve in human and animal nutrition. Over-expression of ferritins in seeds and cereal grains
resulted in an increased Fe content in these edible parts (Goto et al., 1999; Lucca et al., 2002).
However, over-expression in vegetative tissues did not have this effect (Drakakaki et al.,
2000), and in some cases even caused Fe deficiency symptoms (Van Wuytswinkel et al.,
1999). Overall, ferritin over-expression has to studied in more detail and it may be needed to
increase Fe uptake at the same time to have a full effect of Fe increases (Qu le et al., 2005).
Thus, research on the influence of ferritin on Fe accumulation and bio-availability as well as
its effect on human Fe uptake revealed that this protein is a promising candidate for bio-
fortification approaches if utilized in an appropriate manner in plants.

4.3 Increase of nicotianamine content

Nicotianamine is a key compound of metal homeostasis in plants. Nicotianamine is a non-
proteinogenic amino acid derived from S-adenosyl methionine by the action of the enzyme
nicotianamine synthase. Nicotianamine is able to bind a number of different metals
including ferrous and ferric Fe, depending on the pH environment. Nicotianamine ensures
that Fe remains soluble inside the cells. Thus, Fe can be transported to the multiple
compartments, and Fe toxicity effects are reduced. Nicotianamine contributes to all
important sub-processes of plant metal homeostasis: Mobilization and uptake, intercellular-
and intracellular transport, sequestration, storage and detoxification of metals. Several
studies presented positive effects of nicotianamine on Fe uptake and accumulation in seeds
(Cheng et al., 2007; Douchkov et al., 2005; Douchkov et al., 2001; Klatte et al., 2009).
Therefore, nicotianamine can be considered to be a potential bio-fortification factor for Fe in
seeds and grains of crop plants. (Lee et al., 2009) showed that overexpression of a
nicotianamine synthase gene, OsNAS3, resulted in an increase of Fe in leaves and seeds, and
that in seeds a higher nicotianamine-Fe content was present. Moreover, it was found that
these transgenic seeds provided a better source of dietary Fe than the wild type seeds (Lee et
al., 2009). (Zheng et al., 2010) demonstrated by seed-specific expression of OsNAS1 that rice
grains contained a higher amount of nicotianamine. These transgenic rice grains performed
better in Fe utilization studies using human cells (Zheng et al., 2010). Other studies also
indicated that simple overexpression of nicotianamine synthase genes may result in
increased nicotianamine but not necessarily in augmented Fe utilization by the plants
(Cassin et al., 2009). Excessive nicotianamine may restrict the availability of Fe when present
in the apoplast (Cassin et al., 2009). It was also found that nicotianamine synthase
overexpression can result in increased levels of Fe in leaves, but not consequently in seeds.
In conclusion, it can be stated that increased nicotianamine synthase gene expression can
result in beneficial effects on bioavailability of Fe due to the chelator nicotianamine.
However, care has to be taken on the site and amount of expression.
Strategies for Iron Biofortification of Crop Plants 9

4.4 Combination of factors affecting bio-availability of Fe

The above studies suggested that targeting single genes may not necessarily result in an
increased level of bio-available Fe. Combining multiple factors that affect bio-availability can
be of further advantage. Such approaches have been tested. For example, rice grains
expressing Aspergillus phytase, bean ferritin and a metallothionein were produced to contain
higher levels of Fe in a form that might be bio-available (Lucca et al., 2002). In another study,
maize plants were generated that expressed at the same time Aspergillus phytase and soybean
ferritin in the endosperm of kernels (Drakakaki et al., 2005). These plants had an increased Fe
content in seeds by 20-70% and nearly no phytate. Very interestingly, such kernels proved
advantageous in bio-availability studies to human cells (Drakakaki et al., 2005).
(Wirth et al., 2009) produced rice plants simultaneously expressing three transgenes, namely
a bean ferritin gene, Arabidopsis nicotianamine synthase gene AtNAS1 and a phytase.
Combined ferritin and nicotianamine over-production resulted in a stronger increase of Fe
content in the endosperm of grains than was achieved in transgenic approaches with single
genes (Wirth et al., 2009).
Thus, attempts to increase bioavailable Fe in seeds are becoming more successful, and
combining multiple targets for breeding of Fe efficiency and Fe bio-availability seems to be
the key.

4.5 Breeding for novel traits

The above presented approaches rely on the targeting of known components of plant Fe
homeostasis mainly in gene technological approaches. An alternative non-transgenic approach
is to use the genetic pool of germplasm collections to screen for plant lines that are Fe-efficient
and have a high bio-availability of Fe. Such genetic traits can be mapped and backcrossed into
the local elite varieties. An advantage of this genetic screening method is that no assumption
about the physiology of the traits needs to be made beforehand. Due to the power of modern
DNA sequencing the new genes and alleles of interest can eventually be molecularly
identified, such as in the case of a transcription factor gene affecting seed micronutrient
content (Uauy et al., 2006). In these cases, the power of natural genetic variation is utilized
which is based on the natural selection of the best available traits that evolved in the
germplasm collection, frequently based on the interplay of multiple genes and specific alleles
(quantitative traits). As an example, plant breeders have begun screening for mineral content
variation in collections of for example wild wheat (Chatzav et al., 2010), rice (Gregorio et al.,
2000) and bean (Blair et al., 2010). Furthermore, recombinant inbred lines generated from the
original cross of two distantly related inbred lines may help in identifying and mapping of
single and quantitative trait loci, for example in wheat (Peleg et al., 2009) and Medicago
(Sankaran et al., 2009). In a different approach, cellular Fe uptake and bio-availability analyses
have been used to screen rice or maize lines with novel traits not previously associated with
known components of Fe usage (Glahn et al., 2002; Lung'aho et al., 2011).

5. Conclusion
Bio-fortification of crops with micronutrients contributes to the improvement of food quality
and may help reducing the prevalent disease of Fe deficiency anemia world-wide. Multiple
approaches using cereals and other crops have been tested and proven successful. It will
10 Food Quality

remain as a challenge in the future to further improve details of these procedures, e.g. by
exchanging isoforms of the genes, alleles, and new promoters in the case of transgenic
approaches. Genetic breeding approaches can be improved by selecting novel recombinant
inbred lines and new germplasm for testing. In some studies, the newly generated plant
lines have not only been analyzed at plant physiological level for increased Fe content and
gene/transgene activity but also for their capacity to augment Fe bio-availability to human
epithelial cells (Drakakaki et al., 2005; Zheng et al., 2010) or to cure Fe deficiency anemia
(Lee et al., 2009). Such bio-availability studies need to be performed routinely and also used
in screening procedures to provide criteria for selection of the best plant lines.

6. References
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 2

Monitoring Harmful Microalgae by Using

a Molecular Biological Technique
Tomotaka Shiraishi1, Ryoma Kamikawa2,
Yoshihiko Sako3 and Ichiro Imai4
1Wakayama Research Center of Agriculture,
Forestry and Fisheries,
2University of Tsukuba,
3Kyoto University,
4Hokkaido University,

Japan

1. Introduction
In recent years, cultivation of fish and shellfish possesses an important portion for securing
enough seafood all over the world. While, fisheries industry handling fish and shellfish
derived from cultivation in addition to natural seafood are exposed to the danger of mass
mortality of the reared and toxicities of bivalves, sometimes resulting in serious economic
losses and physiological damages by seafood poisoning.
Certain microalgal species have been clearly demonstrated relationships with a mass
mortality of fish and shellfish and certain symptoms of people which are caused by
consumption of seafood contaminated with toxins. Occurrences of paralytic shellfish
poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrheic shellfish poisoning (DSP),
amnesic shellfish poisoning (ASP) and ciguatera fish poisoning (CFP) are caused through a
food chain from toxin-producing microalgae to fish or shellfish (Hallegraeff 1995).
Otherwise, some microalgal species cause a red tide, the name commonly used for the
occurrence of harmful algal blooms (HABs) that result from local or regional accumulation
of a unicellular phytoplankton species and exert a negative effect on the environment
(Anderson 1994; Smayda 1997). Of the 5000 species of extant marine phytoplankton,
approximately 300 algal species can form red tides, and the distribution of these HAB
species is increasing globally. HABs therefore continue to receive attention in coastal regions
all over the world (Hallegraeff 1993).
The canonical method monitoring HABs is that by observation of morphological features
under a light microscope. This method requires labour, time, expert knowledge on
morphologies of microalgae, and technical skills to observe the species-specific
morphological features. In addition, morphology of microalgae is sometimes changed,
depending on the environmental conditions or their growth phases (Imai 2000). Therefore,
identification of HAB species with ambiguous morphology is quite difficult and sometimes
subjective, and henceforth, problematic particularly in genera comprising both toxic and
16 Food Quality

non-toxic species which have similar morphology. The difficulty of monitoring HAB by
light microscopy has indicated necessity of a more objective, rapid and accurate
identification method for HAB species.
In the last decade, to address the above issue, molecular biological techniques have been
developed for monitoring HAB species (Godhe et al. 2002; Sako et al. 2004; Hosoi-Tanabe
and Sako 2005a). Many of such newly developed methods focus on genetic diversity of a
certain gene that does not change in a short term depending on environmental conditions or
the algal growth phase. This implies such molecular biological techniques can distinguish a
HAB species from a morphologically similar but non-toxic species if the two species have
gene sequences different from each other. Additionally, these assays appear to be time-
saving, accurate, simple, and effective for the mass investigation of samples. So, polymerase
chain reaction (PCR) assay, one of representative molecular biological techniques, is an
indispensable tool in the fields regarding HAB-monitoring, since a PCR-based method
allows us to identify or detect HAB cells more objectively even if they have morphology
difficult to be defined their taxonomy under general microscopic methods (Adachi et al.
1994). Further, real-time PCR assay was later developed which allows us not only to detect
and identify HAB species but also to quantify HAB cells (Bowers et al. 2000).
This is especially useful for resting cysts of certain HAB species. Some HAB species have a
resting stage like a seed as one of their life cycles, and resting cysts of many HAB species are
spheroid or ovate and with neither species-specific colour nor ornament (e.g., Alexandrium; ).
In the chapter, we will introduce the principle of real-time PCR itself at first, and
subsequently focus on several applications of the real-time PCR assay which have been
developed to monitor dynamics of HAB species: e.g., neurotoxin-producing dinoflagellate
Alexandrium species, red tide-forming dinoflagellates Karenia mikimotoi and Cochlodinium
polykrikoides, red tide-forming raphidophytes Chattonella species and Heterosigma akashiwo,
and bivalve-specific killer dinoflagellate Heterocapsa circularisquama.
Further, we introduce a method I and my coworkers have recently developed to process a
lot of environmental seawater samples by using a filtration assay and the simplest protocol
of DNA extraction (Shiraishi et al. 2009.). The simple method allows us to investigate many
seawater samples for monitoring HAB species smoothly by using a real-time PCR assay
with HAB species specific oligonucleotide primers and a probe.

2. Principle and application of real-time PCR assay for harmful algal blooms
In 1991, Holland et al. (1991) developed the new method called “Taq man real-time PCR
assay”, which is based on the 5’ to 3’-exonuclease activity of Taq polymerases, for
monitoring the quantity of PCR product in real-time. Subsequently, the method was
improved by Heid et al. (1996). The feature of the real-time PCR assay is requirement of a
fluorogenic oligonucleotide probe in addition to reagents used in general PCR-based assay.
The emission of 6-carboxy-tetramethyl-rhodamine (TAMRA) attached at the 3’-termini of
probes as a quencher dye suppresses that of 6-carboxy-fluorescein (FAM) attached at the 5’-
termini as a reporter dye due to the proximity between the emissions of two dyes.
Describing the mechanisms of quantification briefly, the labeled probes hybridize with
target DNA or PCR products and subsequently are deleted by the exonuclease activity in
Monitoring Harmful Microalgae by Using a Molecular Biological Technique 17

each PCR-cycle, resulting in release of emission of the reporter dye. A fluorometer, which is
generally equipped with a thermal cycler, detects the released emission of the reporter dye
and quantifies the PCR products. Due to the utility, high-sensitivity, and accuracy of
quantification, real-time PCR assay has been applied to development of a method for
monitoring several HABs.
The first application of Taq man real-time PCR assay to HAB species was performed by
Bowers et al. (2000) to quantitatively detect the toxic dinoflagellate Pfiesteria piscicida and its
close relative Pfiesteria shumwayae. Bowers et al. (2000) designed primers-Taq man probe sets
specifically hybridizes 18S rRNA gene of either P. piscicida or P. shumwayae. The real-time
PCR assay using the primers-probe sets demonstrated high specificity even for single cells.
Similar trials were carried out for the toxic dinoflagellate Alexandrium species (Galluzzi et
al., 2004; Hosoi-Tanabe and Sako, 2005; Dyhrman et al. 2006), Karenia brevis (Gray et al.
2003), Pfiesteria spp. (Zhang and Lin, 2005), the naked harmful dinoflagellate Cochlodinium
polykrikoides, Karenia mikimotoi (Kamikawa et al., 2006), and harmful raphidophytes (Handy
et al., 2005; Bowers et al. 2006; Kamikawa et al., 2006).
Especially, Taq man real-time PCR assay was applied to resting cysts of Alexandrium species
in marine sediments (Kamikawa et al. 2005, 2007; Erdner et al. 2011). The cyst densities
calculated by the real-time PCR assay for Alexandrium cysts were almost identical to those
by the canonical method to monitoring the cysts called primulin-staining (Yamaguchi et al.
1995, ; Kamikawa et al. 2007). However, it is notable that the cyst density calculated by the
real-time PCR assay tends to be lower than that by the primulin method when sediment
samples collected from 1-3cm depth were used (Erdner et al. 2011). This difference between
the real-time PCR assay and the primulin method suggests that the real-time PCR assay may
be influenced by cyst condition and viability (Erdner et al. 2011). Otherwise, there are
unknown species that produce resting cysts with the similar morphology and that are
stained with primulin as well.

3. The noxious dinoflagellate Heterocapsa circularisquama

The dinoflagellate Heterocapsa circularisquama is one of the most noxious phytoplankton in
Japanese coastal areas and causes mass mortalities of both natural and cultured bivalves
such as oyster, manila clam and pearl oyster in Japan (Nagai et al., 1996, 2000; Matsuyama,
1999). Blooms of this species have had significant negative impacts on the shellfish
aquaculture especially in western coastal area of Japan (Matsuyama et al., 1997; Tamai,
1999). H. circularisquama was discovered for the first time in Uranouchi Inlet, Kochi
Prefecture, Japan in 1988, and since that time, bloom occurrences have expanded throughout
the western area of Japan (Matsuyama et al., 2001; Imai et al., 2006).
Monitoring the population dynamics of this species is essential for forecast of the red tide
occurrences, and hence, for the mitigation of the damages, following to early
countermeasures. Generalized seasonal occurrence of this species in summer and autumn
could be determined using conventional optical microscopy (Matsuyama et al., 1996;
Nakanishi et al., 1999; Shiraishi et al., 2007). However, precise identification and
enumeration are difficult because this species is rather smaller than other red tide species
(<30 µm), and there are numerous co-occurring dinoflagellates with similar morphology,
implying that it is difficult to distinguish them from H. circularisquama (Horiguchi, 1995;
18 Food Quality

Iwataki et al., 2004; see also Fig. 1). Moreover, definitive identification of this species is
based on morphology of body scales that can only be visualized using transmission electron
microscopy (Horiguchi, 1995).

Fig. 1. Morphology of Heterocapsa circularisquama (left) and Scrippsiella sp. (right). Bar: 10μm.

Shiraishi et al. (2007) reported that it was possible to monitor H. circularisquama using an
indirect fluorescent antibody technique (IFAT). This IFAT method allowed accurate
detection of the cells even at low densities (lower limit, ca. 1 cell L-1). Field studies using this
method allowed the population dynamics of this species to be determined throughout a
year in Uranouchi Inlet, Kochi Prefecture, Japan (Shiraishi et al. 2008.), and during early
spring and later winter in Ago Bay, Mie Prefecture, Japan. Though the method
demonstrated high specificity, individual H. circularisquama cells at lower density become
difficult to be detected by epifluorescent microscopic observation in samples dominated by
similar sized phytoplankton. This means additional treatments and significantly longer
times were required to detect H. circularisquama cells in such samples. Consequently, there is
still an urgent need to develop a simpler, quantitative method for monitoring H.
circularisquama.
Kamikawa et al. (2006) previously reported a real-time PCR identification method of H.
circularisquama. Though it could be used in the field, the assay as described requires a long
and complex DNA extraction processes. Additionally, since target cells in cultures and
seawater samples were collected by centrifugation, which imply that cells might be lost
during the process and that only small sample volumes (50 mL at most) could be readily
processed at a time. The conventional method using filtration was not feasible for
concentrating H. circularisquama cells because most cells were attached and trapped on the
surface of certain filters (Shiraishi et al., 2007). Thus, it was important to develop a simple
technique for cell collection and DNA extraction to apply the H. circularisquama-specific real-
time PCR assay to the field monitoring.
In our previous study, we developed a simple and quantitative monitoring method of H.
circularisquama using a real-time PCR assay (Shiraishi et al. 2009.). The DNA extraction was
performed within a relatively short time by gently filtering the cells down on a filter and
then simply boiling the filter in a buffer. The population dynamics of H. circularisquama in an
inlet revealed by the real-time PCR assay and by the IFAT assay were well consistent with
each other. Because the method was only simply described in the original paper by the
limitation of printing, we introduce the protocol of the simple real-time PCR assay (Shiraishi
Monitoring Harmful Microalgae by Using a Molecular Biological Technique 19

et al. 2009.) in detail in the following sections. This protocol will be helpful for the studies on
many other dinoflagellate species by real-time PCR assay.

4. Materials and methods

4.1 Organisms and culture conditions
The algal strains of H. circularisquama, K. mikimotoi and Skeletonema sp. were obtained from
the National Research Institute of Fisheries and Environment of Inland Sea, Fisheries
Research Agency. Strains of Heterocapsa triquetra and Heterosigma akashiwo were isolated by
G. Nishitani from Maizuru Bay, Kyoto Prefecture, Japan in 1998 and by I. Imai from
Hiroshima Bay, Hiroshima Prefecture, Japan in 1989, respectively. These strains were
cultured at a temperature of 20 ºC on a 14-h light: 10-h dark photo-cycle under an
illumination at 180 µmol photons m-2 s-1 in modified SWM-3 medium (Chen et al., 1969; Imai
et al., 1996).

4.2 Cell collection and DNA extraction

The most effective method of extracting H. circularisquama DNA from cell pellets was
evaluated using six different protocols, the simplest of which was a TE boiling method
modified from the procedure of Kamikawa et al. (2006). For the basic boiling procedure,
either one cell or 100 cells of cultured H. circularisquama cells were collected on the
Nuclepore polycarbonate membrane filters (pore size 3.0 µm) (Whatman, Maidstone, UK)
by filtration, respectively. Extractions at both cell concentrations were done in triplicate to
assess assay variability. The filter was placed in a 1.5-mL microtube without folding, and
then 750 µL of TE buffer (10 mM Tris-HCl: pH 8.0, 1 mM EDTA: pH 8.0) was added. After
the boiling for 10 min in the TE buffer, the filter was immediately removed. The extracted
DNA sample was stored at -60 ºC until the real-time PCR assay was performed. DNA
extraction efficiency using the TE (Tris-HCl/EDTA) boiling method was compared with that
by the modified CTAB (Cetyltrimethylammonium Bromide) method (Zhou et al., 1999;
Kamikawa et al., 2005) and the proteinase K method (Kamikawa et al., 2005), both of which
are commonly used. The same protocol as described above for the TE extractions was
followed.
To choose the most suitable filter for the DNA extraction of H. circularisquama, the cells were
collected on 6 different filters and the DNA extraction was performed on each filter by the
TE boiling method which was found to be the efficient method from the study described
above. Specifically, one cell and 100 cells of cultured H. circularisquama cells were collected
by filtration onto membrane filters composed of either polycarbonate membranes
(Nuclepore, mesh size 3.0 µm), glass-fibers (GF/C, pore size 1.2 µm) (Whatman, Maidstone,
UK), cellulose mixed esters (pore size 3.0 µm) (Millipore, Tokyo, Japan), cellulose acetate
(pore size 3.0 µm) (ADVANTEC, Tokyo, Japan), polytetrafluoroethylene (PTFE) (pore size
3.0 µm) (ADVANTEC, Tokyo, Japan), or hydrophilic polyvinylidene difluoride (PVDF)
(pore size 5.0 µm) (Millipore, Tokyo, Japan), respectively. The DNA sample was stored at -
60 ºC until the real-time PCR assay was performed.
Based on the results of the extraction efficiency tests on the various tests, a standard curves
consisting of eight-fold serial dilutions (104 to 1 cells) of cultured cells were prepared. Each
20 Food Quality

number of the cells was collected on the Nuclepore filter (pore size 3.0 µm) (Whatman,
Maidstone, UK) by filtration. The DNA extraction was performed by the TE boiling method,
and the real-time PCR assay was carried out in triplicate. The standard curve was
constructed based on the correlation between the threshold cycle (Ct value) and the number
of cells.
A major concern when designing a real-time PCR assay for HABs is whether other co-
occurring microalgae adversely affect the amplification efficiency either by introducing
inhibitors or due to cross-reactivity problems. This possibility was explored in an
examination where H. circularisquama cells (104 to 1 cells) were filtered on the Nuclepore
filters (pore size 3.0 µm) at 20 cm Hg with 105 cells each of H. triquetra, H. akashiwo, K.
mikimotoi and Skeletonema sp. which are frequently co-dominated in western coastal areas of
Japan. A previous study also showed that the primers and probe used in this study are
species-specific and do not react DNA from H. triquetra, H. akashiwo or K. mikimotoi
(Kamikawa et al., 2006). The DNA extraction was performed by the TE boiling method, and
the real-time PCR assay was carried out as follows in triplicate. Obtained Ct values at each
number of cells were compared with those of the control experiment where only H.
circularisquama was used.

4.3 Real-time PCR

The primer set and probe used in this study were based on unique species-specific DNA
sites identified by aligning the D1/D2 LSU rDNA sequence of H. circularisquama
(DDBJ/EMBL/GenBank accession number AB049709) with the correponding dinoflagellate
sequences in GenBank. Primers specific to H. circularisquama were HcirF (5’-
GTTTGCCTATGGGTGAGC-3’) and HcirR (5’-CATTGTGTCAGGGAGGAG-3’) and the
probe was HcirTaqMan (5’-FAM-CACCACAAGGTCATGAGGACACA-TAMRA-3’) that
was labeled at the 5’-end with FAM (carboxyfluorescein) and the 3’-end with TAMRA
(carboxytetramethylrhodamine) (Kamikawa et al., 2006).
Thermal cycling was performed with a Rotor-Gene 3000 (Corbett Research, Mortlake,
Australia) in 200-µL PCR tubes of commodity type. PCR was carried out in 25-µL volumes
comprising 1×PCR EX Taq buffer (containing 20mM Mg2+), 200 µM dATP, 200 µM dTTP,
200 µM dGTP, 200 µM dCTP, 0.3 µM forward and reverse primers, 0.4 µM fluorogenic
probe, and 1.25 U of Taq DNA polymerase (Takara EX TaqTM, TaKaRa Bio Inc., Shiga,
Japan). The PCR conditions were as follows according to Kamikawa et al. (2006): one
heating cycle at 95 ºC for 2 min, followed by 45 cycles at 95 ºC for 10 sec and 54 ºC for 30 sec.
The Ct value was calculated as a cycle number that an amplification curve reached at the
most suitable threshold value.

5. Results and discussion

5.1 Development of a DNA extraction method
In order to examine the most efficient method for DNA extraction, three kinds of DNA
extraction methods were subjected to H. circularisquama cells (100 cells and 1 cell) trapped on
the filter. Figure 2 shows obtained Ct values for one and 100 cells by real-time PCR assay
Monitoring Harmful Microalgae by Using a Molecular Biological Technique 21

with each DNA extraction method. For 100 cells, the DNA extracted with the TE boiling
method was as efficient as with the CTAB method and the proteinase K method (t-test, df =
4, p > 0.05). For 1 cell, the DNA extraction efficiency with the TE boiling method was higher
than that with the modified CTAB method (t-test, df = 4, p < 0.05) and similar to that of the
proteinase K method (t-test, df = 4, p > 0.05). Thus, we can consider that the three methods
are similarly efficient for H. circularisquama cells with high density. Given the importance of
detection the HAB species at low density, the TE boiling method appeared to be the most
useful technique for monitoring H. circularisquama by real-time PCR assay. In addition to its
higher detection efficiency, the TE boiling method is more suitable in simplicity, ease of
execution, lower cost, and shorter execution time than the other two methods.

Fig. 2. Comparison of Ct (Threshold cycle) values obtained with three DNA extraction
methods. TE boiling method, CTAB method and proteinase K and SDS method were
subjected to 100 cells (left) and one cell (right) of H. circularisquama on
Nucleporepolycarbonate membrane filters. Ct values were obtained by using the real-time
PCR assay in triplicate. The bars show the standard deviations.

In order to select the filter which would yield the highest and most consistent recovery of
DNA, samples containing either 1 or 100 cells of H. circularisquama cells were filtered onto
six different types of filter. DNA was then extracted using the TE boiling method and
subjected to real-time PCR-amplification. In the case of 100 cells, the real-time PCR assay
successfully amplified H. circularisquama DNA from all the filters with the exception of the
polytetra fluoroethylene (PTFE) membrane filter (Fig. 3). In contrast, for the 1 cell samples,
the qPCR assay failed to reliably amplify the DNA from all the filters with the exception of
the Nuclepore polycarbonate membrane filter, which could be detected in triplicate. Only
one of three DNA samples extracted from either the cellulose acetate or hydrophilic
polyvinylidine difluoride (PVDF) membrane filters were detected. Therefore, we concluded
that the best filter for extraction and detection of H. circularisquama was Nuclepore
polycarbonate membrane filter.
22 Food Quality

Fig. 3. Ct values obtained with TE boiling DNA extraction method for 100 cells (left) and 1
cell (right) collected on six different filters using the real-time PCR assay. 1, Polycarbonate
filter; 2, Glass fiber filter; 3, Cellulose mixed ester filter; 4, Cellulose acetate membrane filter;
5, PTFE membrane filter; 6, Hydrophilic PVDF filter. Asterisks (*) indicate that Ct value
could be obtained from only one of three filters. ND means that Ct value could not be
obtained from all three filters. The bars show the standard deviations.

In a previous study (Kamikawa et al. 2006), H. circularisquama cells were concentrated by

natural gravity filtration. However, it takes whole day for the concentration by natural
filtration. It is not feasible for the routine works for monitoring natural populations. In
addition, concentrating cells by filtration is not feasible for H. circularisquama, because most
cells are attached and trapped on to the surface of any filters examined (Shiraishi et al., 2007).
Otherwise, more amount of seawater for monitoring is more suitable for detecting cell
during period of low cell density, indicating that concentration of cells from seawater is
important for accurate and sensitive detection.
The experiments above demonstrates that cultured cells of H. circularisquama can be
quantitatively recovered and amplified from a single cell gently filtered onto Nucleopore
filters (pore size 3.0 µm) and extracted using the boiling TE method (Fig. 4).

5.2 Validity of the real-time PCR assay

Serial dilutions of vegetative cells (1-104 cells) of H. circularisquama on Nuclepore filters were
prepared, and then, the DNA was extracted by the TE boiling method. The real-time PCR
assay was performed with the DNA samples in triplicates. The standard curve was
constructed based on the mean Ct values and the number of H. circularisquama cells (Fig. 4).
The obtained relationship between Ct values and the number of cells in logarithmic scale was
linear, and the correlation coefficient was significantly high (r2 = 0.997), indicating that the
simple real-time PCR protocol can quantitatively detect H. circularisquama even from one cell.
Monitoring Harmful Microalgae by Using a Molecular Biological Technique 23

Fig. 4. Real-time PCR assay using eight-fold serial dilutions (104 to 1 cell). The result for each
cell number was represented by each symbol shown in the figure.

In order to examine the effects of the existence of other microalgae on the DNA extraction or
subsequent PCR-based quantification, H. circularisquama cells (104 to 1 cells) were collected
together with 105 cells of H. triquetra, H. akashiwo, K. mikimotoi and Skeletonema sp. on the
Nuclepore membrane filters by filtration. The DNA extraction and the real-time PCR assay
were carried out as described above. The standard curve was constructed based on the
mean Ct values and the number of cells (Fig. 5a). At each number of H. circularisquama
examined, there was no significant difference between the Ct value obtained from H.
circularisquama cells in spite of presence or absence of the other microalgae (t-test, df = 4, p >
0.05, Fig. 5b). The correlation between Ct values and the number of cells in logarithmic scale
was linear, and the correlation coefficient was extremely significant (r2 = 0.991, Fig. 5a). It
was confirmed that the DNA extraction and subsequent PCR-based quantification of H.
circularisquama cells were not inhibited even when other microalgae such as H. triquetra, H.
akashiwo, K. mikimotoi and Skeletonema sp. coexist with H. circularisquama.
The constructed standard curve showed linearity (Fig. 4), and the protocol including
concentration of cells, DNA extraction, and the real-time PCR was not inhibited by the
existence of other microalgae even at 105 cells of H. triquetra, H. akashiwo, K. mikimotoi and
Skeletonema sp. (Fig. 5b). It was clearly demonstrated that the presence of closely related
species (e.g., H. triquetra) and/or many other common red tide species did not affect the
efficiency of DNA extraction and subsequent PCR-based quantification of H. circularisquama
cells.
When there are H. circularisquama cells in addition to much higher abundance of similar
sized phytoplankton in the field, the detection of H. circularisquama cells by the canonical
IFAT method with epifluorescent signal is obscured by the presence of numerous other
microalgal cells. Similarly, an underestimation of cell abundance can be occurred when a
large number of particles such as detritus not only inhibit epifluorescence microscopy
24 Food Quality

observation but also blocks the antibody reaction trapped within the detritus, indicating that
the IFAT method is difficult to be applied to sediments and detritus-rich samples. The real-
time PCR assay described in this study appears to be more feasible and practical for
environmental samples than the IFAT method.

Fig. 5. Relationship between Ct values and the log number of cells. a. Standard curve for H.
circularisquama cells constructed from DNA that was extracted from H. circularisquama cells
plus several microalgae. b. Comparison of detection and quantification efficiency between
DNA that was extracted from H. circularisquama cells (closed bars) and from H.
circularisquama cells plus several microalgae (open bars). The bars show standard deviations.

6. Application to environmental samples

The procedure described above was applied to environmental samples in order to monitor
successively H. circularisquama cells in addition to IFAT assay (Shiraishi et al. 2009.). The cell
densities obtained by the real-time PCR assay were almost identical to the results obtained
by the IFAT method. Hence, it was clearly demonstrated that H. circularisquama could be
quantified by this simple real-time PCR assay as sensitively and precisely as the IFAT
method in the field. It is notable that the detection limit of the real-time PCR assay was 1
cell/L: The most sensitive level currently with real-time PCR assay (Shiraishi et al. 2009.). It
Monitoring Harmful Microalgae by Using a Molecular Biological Technique 25

should be also mentioned that the real-time PCR assay sometimes reacted to some
environmental samples which the IFAT assay did not (Shiraishi et al. 2009.). This
incongruence can be explained by that the real-time PCR assay is more sensitive than the
IFAT assay. Otherwise, the real-time PCR assay might react to cell-free DNA derived from
broken, dead cells of H. circularisquama. Since we have no idea which is true, it is better to
use both methods for monitoring H. circularisquama cells in order to grasp precise dynamics
of the HAB species, escaping both underestimation and overestimation.
In this chapter, it has been demonstrated that the real-time PCR assay can be applied to
monitoring various HABs in field waters. If other HABs can be quantified by the same
manner to present method with slight modification, those microalgae would be easily
monitored with the similar procedures of the DNA extraction at the same time. The
conventional methods for monitoring HABs with optical microscopy might be replaced by
the simple real-time PCR assay in the near future, when the costs of machines and reagents
are lowered to become reasonable.
In addition to seawater samples, real-time PCR assay has been applied for the detection of
the cysts of the toxic Alexandrium species from marine sediments (Kamikawa et al., 2005,
2007, Erdner et al. 2011). Furthermore, the PCR method was also used for the detection of
the cells of Alexandrium species in the tissue of mussels (Galluzzi et al., 2005) in order to
investigate the possibility that the HAB cells are propagated to other areas by transport of
bivalves. When H. circularisquama forms temporary cysts in water columns, those temporary
cysts possibly sink down and survive some periods at the surface of the sea bottom. There
are some reports that H. circularisquama could proliferate in water columns of a new area
after the transportation of bivalves which accompany temporary cysts (Honjo et al., 1998;
Honjo and Imada, 1999; Imada et al., 2001). Given the possibility of the temporary cysts as a
seed-population, the detection of H. circularisquama cells is an urgent need from sediments,
and tissues and fecal pellets of bivalves.

7. Acknowledgment
We would like to thank Drs. T. Uchida, M. Yamaguchi, and Y. Matsuyama (the National
Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency),
and G. Nishitani (Tohoku University) for their kind donation of the culture strains of H.
circularisquama, K. mikimotoi and Skeletonema sp. for this study. RK is a research fellow
supported by the Japan Society for Promotion of Sciences (no. 210528). This work wa
supported in part by a grant from the Fishery Agency of Japan.

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I. (2009). Population dynamics of the shellfish-killing dinoflagellate Heterocapsa
circularisquama monitored by an indirect fluorescent antibody technique and a real-
time PCR assay in Uranouchi Inlet, Kochi Prefecture, Japan. In Proceedings of 5th
World Fisheries Congress, 6c_1006_200, TerraPub, Tokyo.
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 3

Species Identification of Food Spoilage and

Pathogenic Bacteria by MALDI-TOF
Mass Fingerprinting
Karola Böhme1, Inmaculada C. Fernández-No1,
Jorge Barros-Velázquez1, Jose M. Gallardo2,
Benito Cañas3 and Pilar Calo-Mata1
1Department of Analytical Chemistry,
Nutrition and Food Science,
School of Veterinary Sciences,
University of Santiago de Compostela, Lugo,
2Department of Food Technology,

Institute for Marine Research (IIM-CSIC), Vigo,

3Department of Analytical Chemistry,

University Complutense of Madrid, Madrid,

Spain

1. Introduction
Food quality and safety is an increasingly important public health issue. Nowadays, the
topics “food quality” and “food safety” are very close and two important issues in the food
sector, due to the globalization of the food supply and the increased complexity of the food
chain. The consumers need to purchase safe products that do not involve any kind of risk
for health. On one hand, the aim of the “food safety” is to avoid health hazards for the
consumer: microbiological hazards, pesticide residues, misuse of food additives and
contaminants, such as chemicals, biological toxins and adulteration. On the other hand,
“food quality” includes all attributes that influence the value of a product for the consumer;
this includes negative attributes such as spoilage, contamination with filth, discoloration,
off-odors and positive attributes such as the origin, color, flavor, texture and processing
method of the food (FAO, 2003).
The contamination of food products with microorganisms presents a problem of global
concern, since the growth and metabolism of microorganisms can cause serious foodborne
intoxications and a rapid spoilage of the food products. Thus, the acceptance and safety of a
food product for the consumers depends in great part on the presence and nature of
microorganisms. Besides molds and yeasts, bacteria are the principle responsible for various
types of food spoilage and foodborne intoxications (Blackburn, 2006). It has to be mentioned
that a food product naturally contains an indigenous microbiota that can include spoilage
and/or pathogenic bacterial species. Depending on the preservation method these species
30 Food Quality

can proliferate and adulterate the product. However, most bacterial contamination occurs
during processing and manipulation of the food products.

1.1 Spoilage bacteria

Spoilage bacteria are microorganisms that cause the deterioration of food and develop
unpleasant odors, tastes, and textures. A spoiled food has lost the original nutritional value,
texture or flavor and can become harmful to people and unsuitable to eat. The microbial
spoilage of food products constitutes an important economic problem, as it results in high
economic losses for the food industry, especially under incorrect refrigeration conditions.
Thus, spoilage bacteria are able to grow in large number in food, decompose the food and
cause changes in the taste/smell, which affect the quality of the products. Spoilage bacteria
normally do not cause illness; however, when consumed in high concentration, they can
cause gastrointestinal disturbance (Blackburn, 2006). There are different bacterial species
that can cause spoilage in food products and the spoilage microbiota depends in great part
on the processing and preservation method. Storage temperature also plays a key role in the
growth of undesirable microbiota in food. Thus, fresh foodstuffs such as fish and meat,
stored at refrigeration temperatures can result in the growth of Pseudomonas spp., including
spoilage species, such as P. fragi and P. putida. A light preservation and change in the
atmosphere, e.g. by vacuum-packaging, may inhibit these bacterial species and favour the
growth of other species, such as lactic acid bacteria (LAB), Enterobacteriaceae, Bacillus spp.
and Clostridium spp. These last two genera are able to produce spores that can survive heat
treatments and germinate after a pasteurization process, being an important issue in food
safety. Spoilage species may be more food-specific and, thus, Erwinia spp. has been reported
in products of vegetal origin. Otherwise, seafood products are commonly spoiled by species
such as Shewanella spp. or Photobacterium spp. In general, bacteria can spoil different foods
depending on the physical-chemical preservation profile (Gram et al., 2002).

1.2 Pathogenic bacteria

Foodborne diseases are caused by agents that enter the body through the ingestion of food.
Food can transmit disease from person to person, as well as serve as a growth medium for
bacteria that can cause food poisoning. The global incidence of foodborne diseases is
difficult to estimate, but it has been reported that in 2005 alone 1.8 million people died from
diarrheal diseases. A great proportion of these cases can be attributed to the consumption of
contaminated food and water. In industrialized countries, the percentage of the population
suffering from foodborne diseases each year has been reported to be up to 30% (WHO,
2007). Pathogenic bacteria often do not change the color, odor, taste or texture of a food
product, being hard to recognize if the product is contaminated. Food-borne infection is
caused by bacteria in food. If bacteria become numerous and the food is eaten, bacteria may
continue to grow in intestines and cause illness. Food intoxication results from consumption
of toxins (or poisons) produced in food as a by-product of bacterial growth and
multiplication in food. In this case the toxins and not bacteria cause illness. Toxins may not
alter the appearance, odor or flavor of food. Common bacteria that produce toxins include
Staphylococcus aureus and Clostridium botulinum. In some cases, such as Clostridium
perfringens, illness is caused by toxins released in the gut, when large numbers of vegetative
cells are eaten. Bacterial food poisoning is commonly caused by bacterial pathogenic species
Species Identification of Food Spoilage
and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting 31

such as Escherichia coli, Salmonella spp., Listeria monocytogenes, S. aureus, Bacillus cereus, C.
perfringens, Campylobacter spp., Shigella spp., Streptococcus spp., Vibrio cholerae, including O1
and non-O1, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia
pseudotuberculosis. Emerging foodborne pathogens may refer to new pathogens, pathogens
that emerge due to changing ecology or changing technology that connects a potential
pathogen with the food chain or emerge de novo by transfer of mobile virulence factors
(Tauxe, 2002). Emerging foodborne pathogens include E. coli O157:H7, Aeromonas hydrophila,
Aeromonas caviae, Aeromonas sobria, Mycobacterium spp., vancomycin-resistant enterococci,
non-gastric Helicobacter spp., Enterobacter sakazakii, non-jejuni/coli species of Campylobacter,
and non-O157 Shiga toxin-producing E. coli.

2. Bacterial identification methods

In order to control and minimize the microbiological hazard of foodborne pathogens, as
well as to predict and enhance shelf-life of food products, pathogenic and spoilage bacteria
need to be identified in a rapid and unequivocal way. Several methods have been designed
to achieve bacterial identification.

2.1 Bacterial identification by classic methods

Traditionally, bacterial species have been identified by classic tools relying on culturing
processes coupled to morphological, physiological, and biochemical characterization.
Phenotypic identification is based on direct comparison of phenotypic characteristics of
unknown bacteria with those of type cultures. The reliability of this kind of identification is
in direct proportion to the number of similar phenotypic characteristics.
When classifying microorganisms, all known characteristics are taken into account.
However, certain characteristics are selected and used for the purpose of identification.
Primary identification usually involves a few simple assays such as colony morphology,
Gram staining, growth conditions, catalase and oxidase tests (Duerden et al., 1998). Testing
the requirements for growth includes the presence or absence of oxygen and the growth
ability on different culture media. For a better approximation of bacterial identification,
other laborious techniques are employed, such as microscopic observation, type of
hemolysis, and biochemical arrays like tests for aminopeptidase, urease, indol, oxido-
fermentation, coagulase test, analysis of resistance to different substances, etc. To make
these analyses faster and less laborious, biochemical assays with multitest galleries are
applied. With these arrays inoculation, incubation and lecture can be carried out on a
minimal space and the whole process can be automated. Using these tests it is usually
possible to characterize the bacterial genus and even species to that an unknown strain more
likely belongs to. Conventional identification methods are widely used despite some
disadvantages. Apart from being slow and laborious, they can be used only for organisms
that can be cultivated in vitro and furthermore, some strains exhibit unique biochemical
characteristics that do not fit into patterns that have been used as a characteristic of any
known genus and species.
In the last decades, the progress of microbiological identification turned to more rapid and
sensitive methods, including antibody-based assays and DNA-based methods, together
with important advances in bioinformatic tools. Thus, some methodologies such as ELISA
32 Food Quality

or PCR already became classic. PCR coupled to sequencing tools has provided a big
amount of information that has been deposited in public databases and is freely available.
Recently, the development of rapid and high sensitive techniques, such as real-time PCR
(RTi-PCR), DNA microarrays and biosensors, provoked the replacement of traditional
culturing methods in the field of bacterial identification in clinical diagnostics, as well as
in the food sector (Feng, 2007; Mohania et al., 2008). Furthermore, Fourier transform
infrared spectroscopy (FT-IR) has been described as a new method for rapid and reliable
bacterial identification (Sandt et al., 2006). At the same time, proteomic tools, such as mass
spectrometry were introduced for the identification of microorganisms (Klaenhammer et
al., 2007).

2.2 Bacterial identification by DNA-based methods

Nowadays, in the field of bacterial diagnostics, traditional culturing methods have been
replaced by molecular techniques based on the analysis of DNA, being much faster, more
sensitive and accurate. However, they usually have a superior cost due to that they
require specific industrial equipment and more qualified personal than conventional
techniques.
The sequencing of the 16S rRNA gene is a common tool for bacterial identification.
Universal primers are designed and bind to conserved regions to amplify variable regions.
The amplification is carried out by means of PCR (Polymerase Chain Reaction). Sequences
of the 16S rRNA gene have been determined for an extremely large number of species and
are accessible on huge DNA databases, such as the GenBank of the National Center for
Biotechnology Information (NCBI). For bacterial identification the 16S rRNA sequence of an
unknown strain is compared to the database of published sequences and the most similar
bacterial strains are determined using the common bioinformatic tool BLAST.
Other DNA genes have also been used for the study of phylogenetic relationships, such as
the 23S rRNA, the intergenic spacer region of 16S-23S (ITS), rpoB (subunit β of RNA
polymerase) and gyrB (subunit β of the DNA girasa). However, for bacterial identification
these genes are not yet applied, due to the lack of amply databases as reference.
More recently, RTi-PCR has been introduced for the detection and quantification of major
foodborne spoilage and/or pathogenic bacteria. As a consequence, a large number of RTi-
PCR procedures are currently available for the specific detection and quantification of
foodborne bacteria, such as Salmonella spp., L. monocytogenes, S. aures or Leuconostoc
mesenteroides (Hein et al., 2001; Malorny et al., 2004; Elizaquível et al., 2008).
The state-of-the-art DNA-based technique is DNA microarray that consists of gene arrays
that can hybridize multiple DNA targets simultaneously, and thus, have enormous potential
for detection and identification of pathogens. Thanks to the increase in the complete
microbial genome sequences, DNA microarrays are becoming a common tool in many areas
of microbial research in the field of bacterial identification in clinical diagnostics, as well as
in the food sector (Severgnini et al., 2011). Microarray is a powerful, sensitive and specific
technology that allows an accurate identification based on single target detection and can
determine subtle differences in the genome.
Species Identification of Food Spoilage
and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting 33

2.3 Mass spectrometry for bacterial identification

In the past few years, proteomic tools, such as mass spectrometry, were introduced for
microbial identification (Klaenhammer et al., 2007). According to the ionization source, mass
spectrometry can be divided in two techniques electrospray ionization (ESI) and matrix-
assisted laser desorption/ ionization (MALDI). The mass analyzer uses an electric or magnetic
field in order to accelerate the ions and produce their separation, by means of the ratio
mass/charge (m/z). In the field of bacterial identification, mass spectrometric methods have a
high potential, due to the ability to detect and identify bacterial proteins (van Baar, 2000).
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF
MS) proved to be a technique with high potential for microorganism identification due to its
rapidness, reduced cost and minimal sample preparation compared to conventional
biochemical and molecular techniques (Seng et al., 2009; Bizzini et al., 2010; Giebel et al.,
2010). With MALDI-TOF MS intact bacterial cells can be analyzed in a rapid way, obtaining
high specific spectral profiles in the low-mass range of 1500 – 20000 Da. The soft ionization
technique allows the analysis of intact high mass molecules, such as proteins. Studies, in
that bacterial cells were treated with trypsin and lysozyme before analysis by MALDI-TOF
MS showed that the spectral patterns detected by this method are generally attributed to
intracellular proteins (Conway et al., 2001). In further studies the proteins detected by
MALDI-TOF MS were identified and resulted that most peaks in the spectral profiles are
derived from ribosomal proteins (Ryzhov and Fenselau, 2001).
Bacterial identification can be carried out by either identifying ion biomarker masses that
could be correlated with theoretically determined protein masses in databases or by
comparing the whole spectral profile to a reference database (van Baar, 2000). In the first
approach, bacterial strains are identified by determining the masses of biomarker proteins
by MALDI-TOF MS and searching against a protein database by matching the masses
against sequence-derived masses (Demirev et al., 2004). A number of studies have been
carried out, determining protein biomarkers by MALDI-TOF MS for bacterial species
identification (Demirev et al., 2004; Fagerquist et al., 2005). A critical challenge of protein
database searches is the necessary high mass accuracy, since some proteins have very
similar masses. Furthermore, identification is limited to well-characterized microorganisms
with known protein sequences available in proteome databases (Dare, 2006).
The second approach, also named “fingerprint approach” is the most applied for bacterial
identification. It relies on spectral differences of bacterial species and identification is carried
out by comparison of the spectral profile of an unknown strain to a reference database of
spectral profiles (Giebel et al., 2010; Mazzeo et al. 2006). This approach allows the
differentiation of bacterial strains, due to the high specific spectral profiles, named
“fingerprints”, obtained. For this purpose, it is not necessary to identify the proteins but just
to determine a number of characteristic peaks that are representative for the corresponding
species and/or genus.

3. MALDI-TOF MS fingerprinting, a rapid and reliable method for bacterial

identification in food
MALDI-TOF MS fingerprinting proved to be applicable for bacterial identification at the
genus-, species- and even strain level. In routine bacterial identification in the clinical sector
34 Food Quality

it demonstrated to be a rapid, cost-effective and accurate technique that achieved correct

species identification of more than 92%. This is a significantly better result than conventional
biochemical systems or even 16S rRNA sequencing for bacterial identification (Seng et al.,
2009; Bizzini et al., 2010). Several authors agree that the costs of bacterial identification by
MALDI-TOF MS fingerprinting is around two-thirds less than conventional methods, when
taking into account the cost of materials and staff (Hsieh et al., 2008; Nassif, 2009).
Furthermore, it has several advantages over other fast methods relying on genomics, such as
DNA-microarrays, because fewer steps are necessary to achieve bacterial identification and
thus, fewer errors are introduced along the analyzing process. Another advantage of
MALDI-TOF mass fingerprinting is the effortless analysis of results, since no extensive data
processing and statistical analysis is required, as it is the case in other rapid methods for
bacterial identification, such as FT-IR and DNA-microarrays.
Until recently, MALDI-TOF MS techniques for the identification and typing of
microorganisms remained confined to research laboratories and to certain species and the
comparison with other species is limited by the accessibility of spectra. In the last years, the
availability of MALDI-TOF MS devices and the reduction of costs, which enabled their use
in either clinical, food or environmental microbiology laboratories, helped to increase the
number of studies for the identification of different food pathogens. Thus, regarding to the
application to routine bacterial identification, MALDI-TOF MS has shown to be a fast,
reliable and cost-effective technique that has the potential to replace and/or complement
conventional phenotypic identification for most bacterial strains isolated in clinical, food or
environmental microbiology laboratories. Some authors conclude that the identification of
microbial isolates by whole-cell mass spectrometry is one of the latest tools, forging a
revolution in microbial diagnostics, with the potential of bringing to an end many of the
time-consuming and man-power-intensive identification procedures that have been used for
decades. However, to increase the reliability of the method, it should be taken into account
that for routine identification of bacterial isolates, correct identification by MALDI-TOF MS
at the species or strain level should be achieved (Bizzini et al., 2010).
In this sense, bacterial differentiation at the species level is not always possible with the
commonly applied methods for bacterial identification. Thus, the analysis of the 16S rRNA
gene resulted to be complicated in some cases due to the high similarity of sequences of
species of the same genus, such as Bacillus spp. and Pseudomonas spp. However, the correct
identification of the corresponding species is of great importance for food safety and quality,
since the pathogenic and spoilage character can vary significantly. With MALDI-TOF MS
fingerprinting a higher discrimination potential has been described, allowing the
differentiation and correct identification of much close bacterial species and even strains of
the same species (Keys et al., 2004; Donohue et al., 2006; Vargha et al., 2006).
In recent years, several reports have shown the feasibility of using MALDI-TOF MS for
identifying microorganisms (Seng et al., 2009; Giebel et al., 2010). The detection and
comparison of protein mass patterns has become a convenient tool for the rapid identification
of bacteria, due to the high specific mass profiles obtained. It has to be mentioned that most
studies of bacterial identification by MALDI-TOF MS fingerprinting are targeted at clinical
diagnostics of bacterial strains associated with human infectious diseases.
In contrast, only few works have been done in the field of microbial food analysis by
MALDI-TOF MS for the identification of foodborne pathogens and/or spoilers. These works
Species Identification of Food Spoilage
and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting 35

included the classification and identification of several widespread pathogens causing food-
borne diseases such as Aeromonas A. hydrophila, Arcobacter spp., Campylobacter spp.,
Clostridium spp., Listeria spp., Salmonella spp., Staphylococcus spp., V. parahaemolyticus, Yersinia
spp., Bacillus spp. and species of the Enterobacteriaceae family (Bernardo et al., 2002; Bright et
al., 2002; Keys et al., 2004; Mandrell et al., 2005; Donohue et al., 2006; Carbonnelle et al.,
2007; Barbuddhe et al., 2008; Grosse-Herrenthey et al., 2008; Hazen et al., 2009; Alispahic et
al., 2010; Ayyadurai et al., 2010; Dubois et al., 2010; Stephan et al., 2010; Stephan et al., 2011).
Furthermore, some studies were aimed at the detection of foodborne pathogens and food
spoilage bacteria, including genera such as Escherichia, Yersinia, Proteus, Morganella,
Salmonella, Staphylococcus, Micrococcus, Lactococcus, Pseudomonas, Leuconostoc and Listeria
(Mazzeo et al., 2006). In further studies an ample spectral library was created, including the
main pathogenic and spoilage bacterial species potentially present in seafood (Böhme et al.,
2010a; Fernández-No et al., 2010; Böhme et al., 2011b). These works included genera, such as
Acinetobacter, Aeromonas, Bacillus, Carnobacterium, Listeria, Pseudomonas, Shewanella,
Staphylococcus, Stenotrophomonas, Vibrio and genera of the Enterobacteriaceae family.
In Figure 1 spectral profiles of some important foodborne pathogens and spoilers are
shown, demonstrating the high specificity. Böhme et al. (2010a, 2011b) also determined
characteristic biomarker peaks for every studied genus and species. Such unique or
characteristic peak masses can serve for the rapid identification of a bacterial genus and/or
species. However, unequivocal identification can not be carried out based on a single
biomarker protein, but under consideration of a number of characteristic mass patterns,
representing the spectral fingerprint.
Furthermore, when working with microbial mixtures, such biomarkers become more
important, since the presence or absence of unique peak patterns could lead to a conclusion of
the present bacterial species. The detection of biomarker proteins by MALDI-TOF MS has been
successfully applied for the identification of two bacterial species isolated from contaminated
water, lettuce and cotton cloth (Holland et al., 2000). However, until now, the application of
MALDI-TOF MS fingerprinting for microbial mixtures has not yet been demonstrated.
Another critical challenge of MALDI-TOF MS fingerprinting is that the classification of a
bacterial genus or species, as well as the determination of unique biomarker patterns, is only
possible in the frame of the content of the spectral reference library. However, the number of
studies on bacterial species identification by MALDI-TOF MS in foodstuffs is continually
increasing and so does the reliability of the identification.
Figure 2 shows the scheme of the whole process of bacterial identification by MALDI-TOF
MS in food products. Bacteria are isolated from food samples and cultivated to obtain single
colonies. Afterwards, the bacterial cells are lysed by an organic solvent and a strong acid,
being the most applied ones Acetonitrile (ACN) and Trifluoracetic acid (TFA). Once
obtained the spectral profiles for each bacterial strain, data analysis is carried out, including
the extraction of representative peak mass lists and the comparison of spectral data, with the
aim of bacterial discrimination. Furthermore, cluster analysis of the peak mass lists reveals
phyloproteomic relationships between bacterial species, allowing the identification of
unknown strains, as well as the typing of closely related strains. For the sample preparation
and data analysis a number of different protocols and techniques have been described and
are discussed in the following sections.
36 Food Quality

Fig. 1. MALDI-TOF MS profiles of some foodborne and spoilage bacteria.

For bacterial identification by MALDI-TOF MS fingerprinting, the spectral profile of the

strain of interest is compared to a spectral library of reference strains. Several private
databases have been created, including spectral profiles of more than 500 bacterial strains,
such as The Spectral Archive And Microbial Identification System (SaramisTM; AnagnosTec
GmbH, Potsdam, Germany) (Erhard et al., 2008) and the Microbelynx bacterial identification
system (Waters Corporation, Manchester, UK) (Keys et al., 2004; Dare, 2006). The MALDI
Biotyper 2.0 (Bruker Daltonics) search against an ample database of more than 1800 bacterial
species and new spectral profiles are being added on a daily basis. The database
demonstrated to be applicable for the routine bacterial identification in the clinical sector,
being a rapid, cost-effective and accurate technique that achieved correct species
identification of 92% (Seng et al., 2009; Bizzini et al., 2010). As already mentioned, most
studies, as well as these databases, are targeted at human pathogens causing infectious
diseases. Nevertheless, the databases also include bacterial species that play an important
role in food safety and quality and could represent important reference data for the
identification of food pathogens and food spoilage bacterial species. However, the critical
challenge of these databases is the limited availability. In this sense, it would be desirable a
public database for the submission of the spectral information for each species that would
allow the comparison with results from different researches and favoring a more precise
method for identification of intact bacteria based on a huge amount of data.
Species Identification of Food Spoilage
and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting 37

Fig. 2. Scheme of the protocol for the identification of foodborne and spoilage bacteria by
MALDI-TOF MS fingerprinting.

Thus, a few attempts to start a public database have been achieved. Mazzeo et al. (2006)
constructed a library containing spectra of 24 food-borne bacterial species, including
Escherichia spp., Yersinia spp., Proteus spp., Morganella spp., Salmonella spp., Staphylococcus spp.,
Micrococcus spp., Lactococcus spp., Pseudomonas spp., Leuconostoc spp. and Listeria spp.
Although, the spectral profiles and peak mass lists are freely available on the Web
(http://bioinformatica.isa.cnr.it/Descr_Bact_Dbase.htm), the library only includes a few
bacterial species important in food-borne diseases and/or food spoilage.
A reference library of mass spectral fingerprints of the main pathogenic and spoilage
bacterial species, potentially present in seafood products has been created, including more
than 50 bacterial species with interest in the food sector (Böhme et al., 2010a; Böhme et al.,
2011b). In further studies, the library showed to be applicable for the correct identification of
unknown bacterial strains isolated from commercial seafood products (Böhme et al., 2011a).
It should be emphasized that the compiled reference library of seafood borne and spoilage
bacterial species can be applied to any other foodstuff. The constituted spectral library may
easily be enlarged by further bacterial species and strains that are of interest in the
corresponding food product.

3.1 Sample preparation protocol

To allow the comparison of bacterial spectral fingerprints, the obtained spectra have to be
representative and reproducible. For that, a standardized protocol has to be followed,
beginning from sample preparation to instrumental parameters. It has been shown that
spectral profiles were less sensitive to culture conditions but can show significant variability
depending on the sample preparation protocol (Bernardo et al., 2002; Wunschel et al., 2005).
38 Food Quality

In the first studies of bacteria by MALDI-TOF MS, protein fractions were isolated from
bacterial cells but shortly turned to the analysis of whole cells directly without any sample
pre-treatment, called intact cell mass spectrometry (ICMS). Many authors focused on the
optimization of the sample preparation protocol, with the aim to establish a standardized
protocol to obtain specific and reproducible spectral profiles in a rapid and labor-saving
way (Williams et al., 2003; Mazzeo et al., 2006; Liu et al., 2007). Nowadays, three different
sample preparation protocols are commonly applied. Table 1 resumes these different
methods, highlighting their advantages and disadvantages. The most rapid and labor-
saving method is based on the direct application of bacterial biomass taken from culture
plates to the MALDI-TOF MS sample plate. Afterwards, the bacterial cells are overlaid with
the matrix solution (Bright et al., 2002). Apart from being the most rapid and labor-saving
method, the direct spotting of biomass to the sample plate had several disadvantages. The
difficulty in taking the correct amount of biomass complicates to get a homogenous
distribution of the sample and matrix. Although, this technique was successfully applied for
bacterial species identification (Keys et al., 2004; Erhard et al., 2008), it has been shown that
spectra showed more noise and less peak resolution with this fast method, making difficult
to obtain reproducible spectral profiles (Böhme et al., 2010b). Another sample preparation
technique analyzed cell suspensions that were obtained after harvesting bacterial biomass in
a solvent, including one or two washing steps and resuspension of the pellet in the matrix
solution (Mazzeo et al., 2006; Vargha et al., 2006). The disadvantages of this method are the
time-consuming washing steps and the loss of small soluble proteins. Some authors also
described a similar sample preparation method, but where no washing step was applied
and the bacterial colonies were harvested in a solvent to obtain cell suspensions
(Carbonnelle et al., 2007).

Whole Cells Whole Cell Suspension Cell extract

Sample preparation
Direct application of Harvest biomass in organic Harvest biomass in
biomass to target well solvent organic solvent
1-2 cycles of Washing/ 1 Centrifugation step
Centrifugation steps Analysis of supernatant
Advantages and disadvantages
Very fast method Time consuming washing steps Very fast method
Less homogenous Homogenous crystallization Homogenous
crystallization crystallization
Less reproducibility Good reproducibility Best reproducibility
More noise More noise Low noise
Less resolution of peaks Good resolution of peaks Best resolution of peaks
Table 1. Comparison of different sample preparation protocols.

In a third approach, based on the latter one, cell suspensions were centrifuged and spectra
obtained by the analysis of the supernatant. When comparing to the spectra resulting from
cell suspensions without centrifugation step, the spectral profiles obtained of the
supernatant showed a better reproducibility, a higher resolution and less noise. The
decreasing of noise, lessening the background, and the increase in resolution leads to more
representative and characteristic peaks for each bacterial species improving the
reproducibility (Böhme et al., 2010b). It should be mentioned that, in general terms, a more
Species Identification of Food Spoilage
and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting 39

homogenous distribution of sample and matrix is expected with cell extracts, than with cell
suspensions. In addition, this sample preparation method is rapid and effortless, since the
extracts were obtained directly from cell cultures in just one dilution/centrifugation step.
Although, when working with the extracts it should be expected to find small, soluble
proteins, spectral profiles showed a high number of peaks, similar or even higher than those
obtained by the analysis of whole cell suspensions. Nowadays, most applications of
MALDI-TOF MS for bacterial identification analyzes bacterial cell extracts, obtained by just
one dilution/ centrifugation step.
As mentioned before, for the direct comparison of spectral profiles, with the aim of bacterial
differentiation, a strict standardized protocol has to be followed. It has been shown that,
when applying the same culture conditions, sample preparation, matrix, organic solvents
and MALDI-TOF MS analyzer, reproducible spectral profiles are obtained (Keys et al., 2004;
Barbuddhe et al., 2008). Although, different protocols can lead to a high variability in the
resulting spectral profiles, it has to be mentioned that some peaks were detected, even if
different protocols were applied. Such characteristic and conserved peaks could serve as
biomarker proteins for the corresponding genus and/or species.

3.2 Data analysis and phyloproteomics

For the identification of an unknown bacterial strain by MALDI-TOF MS fingerprinting, the
spectral profile is compared to a spectral library of reference strains. The existing microbial
identification databases MALDI Biotyper 2.0 (Bruker Daltonics), SaramisTM (AnagnosTec
GmbH) and Microbelynx (Waters Corporation) include an amply library of spectral data. At
the same time, the software for analyzing spectral data and carry out bacterial identification
are incorporated to these databases. Identification of unknown microorganisms is
performed by comparing their individual peak lists to the database. A matching score based
on identified masses and their intensity correlation is generated and used for ranking of the
results. Furthermore, based on the similarity scores dendrograms can be constructed and
principle component analysis can be carried out. The critical challenges of these databases
and software are that both are conditioned to the corresponding instrument and are not
available for other investigators without paying high charges.
Thus, in a number of studies, smaller, “in house” libraries have been constructed. The
difficulty of developing an “in house” database lays in the need for particular algorithms to
analyze or compare obtained spectra and to carry out searches against the constructed
reference library. Peak matching techniques eliminate the subjectivity of visual comparison.
Jarman et al. (2003) developed an automated peak detection algorithm to extract
representative mass ions from a fingerprint and to compare spectra to fingerprints in a
reference library. This algorithm carries out the identification of an unknown spectrum by
calculating a degree of matching and is robust to the variability in the ion intensity (Jarman
et al., 2003). Other authors developed a software (BGP-database, available on
http://sourceforge.net/projects/bgp) to analyze and compare spectral profiles, allowing
the rapid identification. This software determines the best match between the tested strain
and the reference strains of the database, taking into account a possible error of the m/z
value (Carbonnelle et al., 2007).
In further studies, the freely available web-based application SPECLUST (http:
//bioinfo.thep.lu.se/speclust.html) was used to extract representative peak masses and to
40 Food Quality

obtain final peak mass lists for each bacterial strain. Later on, required mass lists can be
compared and common peak masses defined. The web interface calculates the mass
difference between two peaks taken from different peak lists and determines if the two
peaks are identical after taking into account a certain measurement uncertainty (σ) and peak
match score (s) (Alm et al., 2006). The web program resulted very fast, easy to handle, and
could be extended by new spectral mass lists in a simple manner. Although, it was not
possible to search an unknown spectrum directly against the library, comparison of peak
mass lists could be carried out and common peaks determined with the aim of identifying a
spectral profile of an unknown strain. The web-application was successfully applied to
identify pathogenic and spoilage bacterial strains, isolated from commercial seafood
products (Böhme et al., 2011a). In addition, the program allows the rapid determination of
specific biomarker peaks and includes a clustering option.
Further bioinformatics programs, such as Statgraphics Plus 5.1 (Statpoint Technologies, inc.,
Warrenton, USA), offer a variety of functionalities. First, spectral data have to be
transformed to a binary table, indicating the presence (1) and absence (0) of a peak mass.
Afterwards, various algorithms for cluster analysis can be applied, as well as Principal
Component Analysis (Böhme et al., 2011b). In a different study, the BioNumerics 6.0
software (Applied-Maths, Sint-Martens-Latem, Belgium) was used for data analysis and
machine learning for bacterial identification by MALDI-TOF MS (De Bruyne et al., 2011).
Clustering of the spectral data obtained by MALDI-TOF MS represents another approach
for bacterial identification and classification. Conway et al. (2001) introduced the term
“Phyloproteomics” and the clustering of peak mass lists allowed a better visualization of
similarities and differences of spectral comparison. The construction of a dendrogram based
on mass spectral data is a rapid technique to analyze spectral profiles and to visualize
spectral relations by grouping the obtained peak mass lists of bacterial strains. Thus, on one
hand, clustering has been successfully applied for the differentiation and identification of
bacterial strains at the genus and species level (Conway et al., 2001; Wunschel et al., 2005;
Vargha et al., 2006; Carbonnelle et al., 2007; Böhme et al., 2010a; Dubois et al., 2010; Böhme
et al., 2011b). On the other hand, clustering of mass spectral data has been applied as a
typing method for the phyloproteomic study of different strains of the same species, with
the aim to classify the strains (Siegrist et al., 2007; Teramoto et al., 2007). When comparing
the dendrograms representing phyloproteomic relations to the phylogenetic trees, a high
concordance were found by these authors. Since the peak patterns observed by MALDI-TOF
MS are generally attributed to ribosomal proteins (Ryzhov and Fenselau, 2001), the
similarity of the MALDI-TOF MS cluster to phylogenetic trees obtained by the analysis of
ribosomal genes is not surprising (Dubois et al., 2010). However, in comparison to the
sequence analysis of the 16S rRNA gene that is commonly used for phylogenetic studies, the
classification of bacterial strains by MALDI-TOF MS fingerprinting resulted to be more
discriminating. This is important for some genera, such as Bacillus and Pseudomonas, for that
the differentiation at the species level is difficult with 16S rRNA analysis.

4. Conclusion
In order to ensure food safety and quality, the objective of this chapter is to review and
update MALDI-TOF MS-based molecular methods that allow for the early detection and
identification of the main pathogenic and spoilage bacteria. In recent years, molecular
Species Identification of Food Spoilage
and Pathogenic Bacteria by MALDI-TOF Mass Fingerprinting 41

diagnosis has increased in importance, representing an attractive alternative to the

traditional techniques of cultivation and characterization for the rapid detection and
identification of microorganisms in food products that may produce foodborne illnesses.
MALDI-TOF MS fingerprinting proved to be a feasible, rapid and cost-effective technique
for the classification and discrimination of bacterial strains. Besides the high potential in
clinical diagnostics, it demonstrated to be applicable for the identification of unknown
bacterial strains isolated from food samples. In addition, the methodology of MALDI-TOF
MS fingerprinting can be applied to different foodstuff, since bacterial strains are isolated
from the corresponding matrix before analysis and the constituted spectral library may
easily be enlarged by adding bacterial species and strains that are of interest in the
corresponding food product. The vast spectral data can be effectively examined by
searching common peaks masses and cluster analysis. Characteristic peak patterns can be
determined and serve as biomarker proteins for the rapid identification of an unknown
spectrum. The creation of a dendrogram based on phyloproteomic relations, allow for the
typing of closely related strains, extending conventional typing methods. When comparing
phyloproteomic clustering with the classical taxonomy approach based on 16S rRNA gene
analysis, high similarities were observed in the grouping of bacterial strains. However,
genetic analysis does not always permit differentiation between species of the same genus
due to the high similarity of DNA sequences, whereas spectral profiles obtained by MALDI-
TOF analysis can reveal specificities for individual bacterial species. Thus, proteomic tools
and phyloproteomic clustering resulted in more discrimination capability of different
species.
The critical challenge of the MALDI-TOF MS fingerprinting approach for bacterial
identification in food is the limited availability of reference data. A huge public database,
such as the GenBank database of the NCBI, which facilitate the comparison of an unknown
DNA sequence to millions of reference strains, is not available for mass spectral data
obtained by MALDI-TOF MS. In future the main objective should be the publication of the
created spectral libraries, including strains obtained from culture collections, as well as
strains isolated from food samples, to allow other researchers the comparison of their
spectra to the library and thus, a more accurate and rapid identification of bacteria in food.

5. Acknowledgements
This work was funded by project 10PXIB261045PR from Xunta de Galicia and by project
AGL2010-19646 from the Spanish Ministry of Science and Technology. The work of K.
Böhme and I.C. Fernández-No is supported by a “Maria Barbeito” and “Lucas Labrada”
research contract from Xunta de Galicia.

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 4
4

Raman Spectroscopy: A Non-Destructive and

On-Site Tool for Control of Food Quality?
S. Hassing1, K.D. Jernshøj2 and L.S. Christensen3
1Facultyof Engineering, Institute of Technology and Innovation,
University of Southern Denmark,
2Faculty of Science, Department of Biochemistry and Molecular Biology, Celcom,

University of Southern Denmark,

3Kaleido Technology,

Denmark

1. Introduction
In recent years there has been an increasing focus from the consumers on food quality i.e.
unwanted substances such as bacteria, pesticides, drug residues and additives as well as
on food composition including nutritional value, healthy additives, antioxidants and the
contents of selected fatty acids. This is also reflected in an increasing interest for organic food
products. It therefore seems appropriate to develop substance specific, non-destructive and
fast measuring techniques that can be used close to the consumer, for monitoring different
properties of food products.
Raman spectroscopy is an example of a fast, non-destructive and molecule specific technique.
As discussed in section 2, Raman spectroscopy involves illuminating the sample with laser
radiation with wavelengths either in the near-infrared (NIR), visible or ultraviolet (UV)
regions, monitoring the light reflected from the sample and analyzing the intensities as a
function of wavelength.
The focus of the chapter is to discuss the applicability of Raman spectroscopy as a
non-destructive and molecule specific tool for monitoring food quality. This goal is achieved
through a discussion of the basic properties of Raman scattering (RS) and experimental
aspects, followed by a discussion of three case studies: 1. Revelation of a pork content in
minced lamb products, 2. Detection and classification of nearly identical anti-oxidants and
3. Detection of pesticides on fruits and vegetables using surface enhanced Raman scattering
(SERS).
In general the requirements to any experimental method suitable for an on-site evaluation of
food quality are:

1. robust and easy to use instrumentation 6. a qualitative and quantitative

2. portable instrument determination of chemical constituents
3. non-destructive measurements 7. a high molecular specificity
4. no or a minimum of sample preparation 8. a measurement of low concentrations of
5. a fast acquisition time unwanted contents
48
2 Food Quality
Will-be-set-by-IN-TECH

Several of these requirements can be met by optical techniques based on some kind of
reflection measurement. The importance of the different requirements will depend on the
specific application, e.g. are the development and implementation of the technique highly
dependant on, whether the commodity should be controlled for an unwanted or wanted
content. Both types of content place requirements on the molecular specificity, however, in
the case of detecting an unwanted content, the concentration is often very low as well.
Figure 1 illustrates the contents of information obtained from three different kinds of reflection
measurements performed on the same green leaf.

Fig. 1. Optical reflections with different information content from a green leaf. Left: Diffuse
reflection (multiple scattering), middle: Diffuse reflection and imaging and right: Molecular
reflection (Raman scattering).
The middle part of the figure shows an image obtained with an optical microscope, magnified
400 times, where the cells containing chlorophyll are resolved. This experimental method is
based on diffuse reflection of white light and imaging and no quantification of the spectral
information is made, except the information visible to the human eye. To the left is shown a
diffuse reflection spectrum of the same leaf (red curve) obtained with a Perkin Elmer, λ900
spectrophotometer equipped with an integrating sphere. The integrating sphere collects
all the light reflected from the leaf enabling an absolute measurement of the reflectance
coefficient. Reflection measurements performed on different kind of green plants and on
different plant parts are similar and contain almost identical spectral information. The
example in figure 1 shows reflection spectra from two types of leaves of a Hibiscus, rosa
sinensis, namely a foliage leaf and a sepal Jernshøj & Hassing (2009). The spectra shows
differences in the absolute reflection values, but are very similar with respect to spectral
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information. The similarity is partly due to a blurring of the molecular signal caused by the
molecular interaction with the surroundings and partly, since the molecular signal in itself
is a composite signal reflecting both the motions of the electrons and nuclei in the molecule.
A closer study shows a higher concentration of secondary pigments, e.g. carotenes, in the
sepals than in the green leaves. A quantitative analysis of chlorophyll and carotenoid from
these spectra is possible, when applying empirical models, this reflects the complex scattering
and interaction processes taking place in the leaf Jernshøj & Hassing (2009); Kortüm (1969).
However, due to the poorly resolved spectra, it may be impossible to discriminate between
the presence of closely related molecular species, such as the antioxidants α- and β-carotene.
The right part of figure 1 shows a Raman spectrum obtained from the same foliage leaf.
As opposed to the diffuse reflection measurements, the application of a laser results in the
generation of a molecular reflection signal with measurable intensity, namely the Raman
scattered light. As clearly seen from the figure, the spectral information is increased
dramatically in the Raman spectrum. As discussed in the next section, the spectral distribution
observed in the spectrum primarily reflects the vibrational motion of the nuclei in the "naked"
molecules.
Summarizing: The outcome of an optical reflection measurement may be compared to a
bar code, which is a well known component in different industries, where different and
often high information content is encoded into this code and placed on a commodity. The
information is read out by measuring the reflected laser light from the bar code, an example
being the price scanner used in supermarkets. The difference between this bar code and
the molecular information, is that the "molecular bar codes" are native parts of the sample,
which are basically determined by the molecular composition. The informational quality of
the particular "molecular bar code" obtainable is defined by the type of interrogative process
used, e.g. imaging, diffuse reflection or Raman scattering.

2. Raman spectroscopy
The section gives an introduction to Raman scattering and point to the potential inherently
present in the Raman effect with respect to obtain detailed molecular information. The section
focuses on the theoretical and experimental challenges that have to be overcome in order to
make different kinds of Raman techniques valuable diagnostic tools in the analysis of food
quality.
Raman spectroscopy involves illuminating the sample with laser radiation with wavelengths
either in the NIR, visible or UV regions, which excites the constituent molecules within the
sample to vibrate. A vibrational Raman spectrum of the molecules is obtained by collecting
the in-elastically scattered light. Each molecule present in the sample has a characteristic set
of nuclear vibrations and thus the sample as a whole has a unique vibrational signature, i.e.
a "molecular bar code" with a high information content. Raman spectroscopy is a class of
well-documented, non-destructive, optical techniques with a high spectral resolution all of
which are based on the Raman effect discovered by C. V. Raman in 1928 Raman & Krishnan
(1928). Today more than 25 different Raman spectroscopies are known Long (2002).

2.1 An experimental view on Raman spectroscopy

Raman spectra can be obtained as reflectance measurements, which means that samples can
be investigated with no or very little sample preparation and as opposed to other widely
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applied optical techniques, such as NIR and Fourier Transformed Infrared (FT-IR), Raman
measurements are not influenced by the presence of water and therefore biological samples
can be measured in their natural environment. Besides, the samples are not influenced by the
measurements and the same samples can be investigated over time, which is essential, when
measuring on food samples. Since the spectral information contained in a non-resonance
Raman spectrum (vide infra) is virtually independent of the laser wavelength and since
a complete Raman spectrum, typically 0 - 3500 cm−1 , only covers a wavelength region
of approximately 100 nm (the exact value depends on the specific laser wavelength), it
follows that complete Raman spectra of food products can be measured without removing
the protective film covering the products, just by choosing a laser wavelength that matches
the optical window of the protective film.
Raman spectrometers may be divided into two classes: Dispersive instruments and FT-Raman
instruments. Any dispersive Raman spectrometer consists essentially of four components, a
filter to block the Rayleigh scattered light, an entrance slit (often defined by an optical fiber), a
transmission or reflection grating, where in the latter case the focusing optics is built into the
grating and a CCD detector, which is coupled to a computer. The image of the illuminated
entrance slit or fibre core is formed on the CCD and the different wavelengths contained in
the Raman signal is converted by the grating into different positions on the CCD. Because of
the simplicity of the basic Raman spectrometer, it is possible to build different editions for
different purposes.
Figure 2a shows a typical Raman spectrometer, suited for scientific purposes. The setup,
which is developed at The Molecular Sensing Engineering group, Faculty of Engineering,
Institute of Technology and Innovation, University of Southern Denmark, has been built
having a high degree of flexibility in mind. This flexibility allows us to arrange and
rearrange the setup according to the experimental conditions necessary to achieve the desired
molecular information. One of the main research areas is molecular investigations on
bio-molecules, such as porphyrin and haemoglobin doing resonance and non-resonance
Raman spectroscopy, polarized and unpolarized experiments as well as Surface Enhanced
Resonance Raman Scattering (SE(R)RS) Jernshøj & Hassing (2010). Especially research
involving polarized Raman measurements, where the molecular information obtained using
linearly or circularly polarized light, has been carried out on a number of different samples.
Recently, the molecular information obtained from such polarized measurements on a highly
symmetric gold nanostructure (SE(R)RS) has been investigated in details K. D. Jernshøj &
Krohne-Nielsen (2011).
When combining a Raman spectrometer with an optical microscope, the information content
may be further increased. The Raman setup in figure 2a consists of a modified Olympus
BX60F5 microscope, a SpectraPro 2500i spectrograph from Acton (Gratings: 1200 and 600
lines/mm) and a cooled CCD detector from Princeton Instruments, model Acton PIXIS. The
setup is equipped with 12 different laser excitation wavelength, provided by: a 532 nm
diode laser (Ventus LP 532), a Spectra Physics 632.8 nm HeNe laser, a Spectra Physics Ar+
laser: visible region and a tunable Ti3+:sapphire (titanium sapphire) laser: visible and NIR
region. The setup has adjustable spectral, 5cm−1 and spatial resolutions, 0.3μm. Since the
Raman spectrometer is combined with an optical microscope, equipped with a motorized,
translational XY-stage (Thorlabs Inc., CRM 1) on the microscope translational stage, it is
possible to obtain complete Raman spectra from different points across the sample. Due to
the high spatial resolution (0.3μm) it is possible to perform Raman Imaging with a subcellular
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Fig. 2. The Raman equipment at The Molecular Sensing Engineering group at Faculty of
Engineering, Institute of Technology and Innovation, University of Southern Denmark. (a.)
The Raman setup, which is suited for scientific purposes. The Raman spectrometer is
combined with an optical microscope, which is equipped with a motorized XY-stage on the
translational stage. (b.) A portable commercial Raman setup: DeltaNu Inspector Raman, 785
nm excitation, unpolarized Raman, spectral resolution: 10, 12 or 15 cm−1 , predefined laser
power 1.8, 3.4 and 6 mW, polystyrene standard for wavelength calibration, battery operated.
Inspector Raman has been equipped with a manual translational XY-stage, order to be able to
translate the laser across the sample.

spatial resolution, which has proven especially useful combined with multivariate analysis
in the study related to early diagnosis of human breast cancer cells Martin Hedegaard &
Popp (2010). As discussed in case study 3.1, Raman imaging combined with a machine vision
system can be applied in an automatized determination of the composition of inhomogeneous
food products, such as minced meat products.
For the purpose of an on-site, non-destructive investigation, the laboratory is also equipped
with a commercial portable Raman spectrometer, which is shown in figure 2b. The parameters
of this turn-key instrument are: 785 nm excitation, unpolarized Raman measurements,
predefined spectral resolutions: 10, 12 or 15 cm−1 , predefined laser power 1.8, 3.4 and 6
mW, polystyrene standard for wavelength calibration and finally the possibility for battery
operation. According to requirement 2 on page 2, dealing with portability, this kind of small
Raman spectrometer is suited for field use, since it may be battery operated and is portable.
Besides, the spectrometer has been equipped with a manual XY-stage in order to be able to
scan the laser across the samples, when measurements are done on inhomogeneous samples.
As will be discussed in the next section, laser induced fluorescence, which is excited
simultaneously with the Raman process, may often be a serious problem in practical
applications of Raman spectroscopy. The fluorescence can be avoided in most cases by
choosing the laser wavelength in the NIR region. When this choice is combined with an
instrument that has a high sensitivity (high through-put), Raman spectra of high quality can
be obtained. In FT-Raman spectrometers the grating is replaced by a scanning interferometer
(e.g a Michelson interferometer) by which an interferogram, i.e. a time signal containing the
spectral distribution of the Raman signal, is measured. The Raman signal is calculated by
a computer by performing a fast Fourier transform of the interferogram data. A FT-Raman
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spectrometer consists of the following components: a NIR-laser with wavelength 1064 nm,
filters to block the Rayleigh scattered light, a stable and efficient interferometer, a sensitive
detector coupled to a computer, which includes software with the capability of performing
fast Fourier transform. Typically, FT spectrometers are scientific instruments but it is possible
to buy a FT-Raman spectrometer, e.g. RamanPro, which is designed to measure chemicals in
production environments (http://www.rta.biz/).
Since different portable, commercial Raman instruments with different specifications are
available, it will be possible to optimize the application with respect to the specific goal by
applying e.g. either multivariate analysis, some form of enhancement of the Raman signal and
specially developed additional software. Notice, that in the case of FT-Raman instruments the
above mentioned flexibility with respect to choosing the laser wavelength is limited.

2.2 Fragments of Raman theory

As mentioned previously Raman spectroscopy involves illumination of the sample with laser
radiation with wavelengths either in the NIR, visible or UV regions. According to quantum
mechanics the intensity of a laser beam is proportional to the number of photons and the
photon energy Long (1969). In molecular spectroscopy it is custom to measure the molecular
and photon energy in the unit cm−1 , where x cm−1 = 107 /y nm. This means that the photon
energy of a NIR laser with wavelength y = 785 nm is equal to x = 12739 cm−1 , while the photon
energy for a visible laser with wavelength 532 nm is 18797 cm−1 . Thus, the photon energy is
inversely proportional to the wavelength.
When the wavelength of the laser is chosen in the spectral region, where the molecules in
the sample do not absorb any light, a fraction of the laser light is scattered. Most of the
scattered light will deviate from the incident laser light only in the direction of propagation,
but will have the same wavelength as the laser. This scattering process is known as Rayleigh
scattering. A small fraction of the scattered light, namely the Raman scattered light, will in
the scattering process also be shifted in wavelength. These measurable shifts are determined
by the physical properties of the scattering molecule and they appear in the Raman spectrum
as characteristic sharply defined peaks as seen in figure 1. Since each molecule gives rise to a
characteristic and unique Raman spectrum, we are presented with a "molecular bar code" with
a high information content. In vibrational Raman spectroscopy, the spectra reflect that each
molecule has a characteristic set of nuclear vibrations. Besides, being shifted in wavelength
the polarization of the Raman scattered light may also be different from the polarization of
the laser light. The shift in polarization is determined by the nature of the various nuclear
vibrational motions, which are also determined by the molecular properties. The quality of
the information contained in the "molecular bar code" can therefore be increased by including
the shift in polarization in the analysis.
In the following we focus on some of the basic principles describing the Raman process,
which are necessary to illustrate the potential of and difficulties with Raman spectroscopy,
when applied in food analysis. The fundamental theory, various aspects and applications of
Raman scattering have been extensively discussed in the literature and we refer to these for
further details eds. R. J. H. Clarke & Hester (n.d.); Long (2002); McCreery (2000); Mortensen
& Hassing (1980); Plazek (1934); Smith & Dent (2005).
Figure 3 gives a schematic overview of the Raman process (left) and the fluorescence process
(right) and the basic expression for the Raman intensity IRaman is given in Eq.(1).
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Fig. 3. Left: The Raman process is a coherent absorption-emission sequence. Right:

Fluorescence is a real absorption followed by a spontaneous emission process, i.e. an
incoherent absorption-emission sequence.

 2
  gv|ρ|ev ev |σ| g0  gv|σ|ev ev |ρ| g0 

IRaman ∝ 4
ν̃Raman ∑ +  · Ilaser (1)
e,v ν̃ev ,g0 − ν̃laser − iγe,v ν̃ev ,gv + ν̃laser + iγe,v 

The horizontal bars represent the energy of the molecular states, 0 is the vibrational ground
state (indicating that none of the molecular vibrations are excited) and v is the final vibrational
state. | g, 0 and | g, v are the symbols for the initial and final molecular states participating in
the Raman process, where g denotes the electronic ground state and 0 and v the vibrational
substates of the initial and final states. |e, v  denotes an electronic excited state in the molecule
and its vibrational substate. ρ, σ = x, y, z, where e · ρ is the electric dipole moment (e is the
charge of an electron). Since each molecule has a characteristic set of nuclear vibrations, v
and v are really sets of numbers, where the value of each number describes the degree of
excitation of each kind of vibrational motion. Thus, v = v1 , v2 , .....vk , ....v3N −6 , where N is the
number of nuclei in the molecule. In the benzene molecule e.g., where N = 12, the set will
consist of thirty numbers, corresponding to the thirty different kind of vibrations that may be
excited.
The Raman process can be thought of as a coherent absorption - emission sequence, in which
the absorption of the incoming laser light is followed by an immediate re-emission of the
Raman scattered light. During the initial absorption the molecule shifts state from | g, 0
to |e, v , while during the re-emission the molecule shifts state from |e, v  to | g, v. In
non-resonance Raman Scattering, where the photon energy of the laser (illustrated by the
red arrow in figure 3) is small compared to the energy of any electronically excited state,
all molecular states, |e, v , contribute to the scattering process and thereby to the intensity
of a particular Raman line in the spectrum. This is reflected through the appearance of a
summation over all molecular states of the molecule in the theoretical expression for the
Raman scattered intensity given in Eq.(1).
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For comparison, the fluorescence process is illustrated to the right in figure 3. This process
is an incoherent absorption - emission sequence, which consists of a genuine absorption of
incident light followed by a genuine emission of light. As illustrated in the figure, the
initially excited molecule is allowed to shift state before it spontaneously emits light, which
destroys the coherent nature of the sequence. When focussing on the emission spectrum
(i.e. the fluorescence), the molecule may during the emission end up in different vibrational
substates. Since the contributions from all the possible transitions have to be added together
in the expression for the emission intensity and since the number of vibrational motions,
3N − 6, may be very large for molecules typically appearing in food products, the different
contributions to the intensity overlap with the result that the spectral distribution in the
fluorescence spectra becomes broad and without much structure. This decreases the quality of
the "molecular bar code" related to fluorescence. Depending on the experimental conditions,
both the Raman and fluorescence processes may be initiated simultaneously. This is illustrated
in the Raman spectrum of the green leaf in figure 1, where the Raman spectrum "rides" on
top of a broad fluorescence background. The fluorescence may even be so pronounced that
the Raman spectrum is partly or completely hidden. One way to avoid the excitation of
fluorescence is to choose a laser with a photon energy smaller than any electronic excitation
energy of the molecules.
It follows from the expression for the Raman intensity given in Eq.(1) that the intensity is
proportional to the intensity of the laser and proportional to the fourth power of the photon
energy of the Raman scattered light. The energy of the final molecular state termed ν̃Raman shi f t
is equal to ν̃Raman shi f t = ν̃laser − ν̃Raman . In Raman spectroscopy one measures IRaman as
a function of ν̃Raman shi f t , see figure 1. Since ν̃Raman shi f t is equal to a vibrational energy
of the molecule, the Raman spectrum depicts the characteristic vibrations of the molecule.
Vibrational energies are typically much smaller than the excited electronic energies of a
molecule, this means that the Raman intensity is approximately proportional to the fourth
power of the photon energy of the laser. Experience shows that the fluorescence may in most
cases be avoided by choosing a NIR laser with wavelength 1064 nm, which corresponds to
the photon energy: 9399 cm−1 . Comparing the latter with the photon energy of the visible
laser with wavelength 532 nm, the Raman intensity is reduced by a factor 16. The loss of
Raman intensity is in the FT-Raman spectrometers mainly compensated for by the removal
of fluorescence combined with the high sensitivity that can be obtained in these instruments
Vidi (2003).
The central part of the expression is the absolute square of the molecular polarizability, αρσ ,
where αρσ describes the change in the electron distribution of the molecule in response to
the interaction with the incoming laser light. Each numerator in αρσ contains a product of
two terms, where each term is called the transition dipole moment, reflecting the transitions
taking place in the molecule during the scattering process. The magnitude and sign of these
in combination with the magnitude and sign of the denominator determine the contribution
from a specific molecular state to the intensity of a particular Raman line. The appearance of
the absolute square of the sum of these contributions reflects the coherent nature of the Raman
process. In fluorescence both the absorption and the re-emission processes are independent
and determined by the absolute square of each transition moment. In fact, the higher information
content observed in Raman spectroscopy has origin in the coherent nature of the Raman process.
The real part of the denominator in the first term, called the resonance term, contains the
energy difference between the energy of the excited state |e, v  and the photon energy of
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the laser, ν̃ev ,g0 − ν̃laser . The imaginary term reflects the energy broadening of the state
|e, v . If the photon energy of the laser is chosen equal to ν̃ev ,g0 , the contribution from this
state to the Raman process becomes dominating causing the Raman signal to be enhanced.
The enhancement depends on the value of the imaginary term but the enhancement may
typically be of the order of magnitude 104 to 106 . The phenomenon is termed Resonance
Raman Scattering (RRS) and is illustrated with the green arrow in figure 3. In practice
the molecular states close to the resonant state will give the largest contribution to Raman
scattering. In RRS it is possible to get information about the participating excited states,
whereas in non-resonance this information is smeared out.
A large bio-molecule, e.g. present in various food products, has a large number of
characteristic vibrations. Although, not all of them are seen in the Raman spectra, the spectra
may often be very complex, i.e. the "molecular bar code" contains a lot of information. Raman
scattering can be performed in several ways with the result that specific parts of the obtainable
molecular information may be reached, i.e. distinct parts of the "molecular bar code" is read
out. Resonance enhancement of the Raman signal can be utilized to obtain information about
specific parts of a large molecule, e.g. if the molecule contains a chromophore, it is possible
to select the laser wavelength close to an electronic absorption of the chromophore with the
result that only those vibrations involving the nuclei of the chromophore are enhanced and
therefore seen in the Raman spectrum. A chromophore is the part of a molecule responsible
for its color, where the color arises, when the molecule absorbs certain wavelengths of visible
light and transmits or reflects others.
A special kind of resonance Raman spectroscopy, termed Raman Dispersion Spectroscopy
(RADIS) Mortensen (1981), involves a quantitative comparison of Raman spectra measured
with a few different excitation wavelengths close to the electronic resonance of the
chromophore. The 3D graph in figure 4 shows the possibilities with RADIS. For each
excitation wavelength the corresponding Raman intensity can be followed as a function of the
Raman shift, while when choosing a specific Raman shift, the intensity of this can be followed
as a function of the excitation wavelength. Due to the narrow line width of the Raman spectra
and the coherent nature of the Raman process each Raman band has its own and distinct
excitation spectrum (termed an excitation profile).
As discussed in subsection 3.2 this enables discrimination between molecularly almost
identical constituents, such as α- and β-carotene even when the amount of one of the
constituent is very small compared to the other.

2.3 Improving Raman sensitivity by nanotechnology

Raman spectroscopy has two serious limitations: First, Raman scattering is inherently a weak
effect (typically 108 incoming laser photons only generate 1 Raman photon) and secondly,
fluorescence is often emitted concurrently with the Raman scattering. Since the fluorescence
signal is typically 4 to 8 orders magnitude larger than the Raman signal, this will be hidden
in the fluorescence background. The fluorescence may stem from the molecules under
investigation or from other molecules in the sample. The latter situation may often arise when
measuring on food samples, where the sample preparation is absent or kept at a minimum.
The weakness of the Raman signal may be improved in different ways. (1): by increasing
the photon energy of the laser (i.e. by choosing a laser with shorter wavelength) so that it
corresponds to a resonance region of the molecule, (2): by improving the signal to noise ratio
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Fig. 4. RADIS, Raman spectra measured with a few different excitation wavelengths close to
the electronic resonance of the chromophore.

through the application of a sensitive and cooled CCD detector and (3): by the application of
advanced signal processing and multivariate methods. Although, a resonance enhancement
of the Raman signal can be achieved by increasing the photon energy of the laser, the amount
of absorption is also increased, which may lead to photoinduced degradation of the sample.
Since in general the amount of fluorescence increases with shorter wavelength, the resonance
enhanced Raman signal may still be partly hidden in the fluorescence background.
On the other hand, when the sample is exposed to a laser with longer wavelength the
fluorescence decreases, but unfortunately, this also leads to a lower Raman intensity through
4
the ν̃Raman dependence shown in Eq.(1). Even though the choice of a longer laser wavelength
may result in an acceptable Raman signal, it would be advantageous to enhance the Raman
signal and in particular relative to the fluorescence.
This may be achieved in Surface Enhanced Raman Spectroscopy (SERS). The enhancement
of the Raman signal may occur, when the scattering molecules are either physisorbed
or chemisorbed to a nanostructured metallic surface often made of gold (Au) or silver
(Ag). Although a large enhancement can be achieved (up to 1014 has been reported), the
introduction of a nanostructured surface will in general influence the Raman signal from the
native molecules and make the scattering process more complex, so that the interpretation
and implementation of SERS require a detailed analysis of the system under investigation.
When a bare nanostructured metal surface is illuminated by the laser, the laser photons
interact with the electrons in the surface layer. When the metal and the adsorbed molecules
are exposed to laser radiation the incoming laser photons interact with the combined system
with the overall result that the intensity of the Raman scattered light is enhanced relative
to the intensity of the Raman signal of the free molecules and relative to the fluorescence.
The interaction between the incoming light, the metal and the adsorbed molecules depends
in a complicated way on the surface morphology, the kind of metal and on the molecule in
question. A detailed discussion of the theory and the various implications of SERS can be
found in the literature Jernshøj & Hassing (2010); K. Kneipp & (Eds.); Ru & Etchegoin (2009);
Willets & Duyne (n.d.). Below, only a brief discussion of SERS is given. Figure 5 illustrates
Raman scattering of molecules adsorbed to a nano-structured metal surface.
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Fig. 5. A simplified schematic representation of SERS. The insert is obtained from R. L.

Eriksen and O. Albrektsen (Faculty of Engineering, Institute of Technology and Innovation,
University of Southern Denmark). Similar structures can be found in reference R. L. Eriksen
& Albrektsen (2010).

A focused laser beam illuminates the molecules adsorbed to the Au or Ag surface. The
diameter of the laser beam is typically 0.6 μm and the size of the nano-structure is 12 - 600
nm. The insert shows a SEM-micrograph of a real nano-substrate based on a Si-substrate with
100 nm spheres coated with a 40 nm thick layer of Au. Due to the interaction between the
laser light and the electrons in the metal surface a local electric field Elocal is created outside
the metal and very close to the surface. Depending on the specific surface structure Elocal
may be much larger than the incoming electric field, Ein , associated with the photons in the
laser beam and Elocal may be rather different in different points on the surface. Surface points,
where Elocal is very high, are called hot-spots. As indicated Elocal will decrease exponentially
with the distance from the surface and only those molecules that are within approximately
10 nm will be influenced significantly by this field. According to electromagnetic theory, the
intensity of a light wave is proportional to the absolute square of the electric field associated
with the wave. As indicated in the figure, the incoming laser light gives rise to ordinary
Raman scattering by the molecules, which are not close to the surface. The Raman intensity of
these molecules is proportional to | Ein |2 . The molecules adsorbed at the surface interact with
the local field and with the electrons in the metal. The result is that both the absorption and the
emission parts of the Raman process become proportional to | Elocal |2 , so that the SERS-signal
becomes proportional to | Elocal |4 . The enhancement of the SERS signal is therefore given by
the factor | EElocal
in
|4 .
Thus in cases, where the local field is just 10Ein , the enhancement of the Raman signal
becomes equal to 10000. For comparison, the fluorescence signal is only enhanced with the
factor | Elocal /Ein |2 . The difference between the enhancement of the Raman signal and
the fluorescence may be attributed to the difference in the coherence properties of the two
processes.
SERS can be performed by using nano-substrates with an ordered structure of the SERS-active
sites. These can be designed and fabricated with specific applications in mind, including
functionalization of the surface with a layer of molecules, which bind reversibly to the specific
molecules to be investigated. There are a large variety of commercial nano-substrates available
on the market. Another possibility is to form metal colloids and mix the samples with the
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colloid solution or by coating e.g. a SiO2 surface with Au aggregates. Colloidal Au has e.g.
been used to chemically identify important components in plant material such as green tea
leaves, shredded carrots or shredded red cabbage Zeiri (2007). SERS active substrates made of
aggregated Au nanoparticles on a SiO2 substrates have been applied to detect single cells of
different bacteria, which are very important in relation to food products, i.e. Escherichia coli
and Salmonella typhimurium W. R. Premasiri & Ziegler (2005). Although Au and Ag based
substrates or colloids are the most commonly used, other materials have been applied. Thus,
the SERS signal from single cells of Escherichia Coli bacteria has been obtained by mixing
ZnO nanoparticles with the bacteria cells R. K. Dutta & Pandey (2009).

2.4 Extracting information from "molecular bar codes" by multivariate analysis

Multivariate analysis is in general applied in order to analyze experimental data by the
use of mathematical and statistical methods. The application of multivariate analysis
to spectroscopic data has indeed become very important for describing small differences
between chemical constituents in samples containing bio-molecules. Spectroscopic data are
often analyzed by using Principal Component Analysis (PCA), to which a brief introduction
will be given in the following, a thorough explanation of this and other multivariate methods
can be found in reference A. K. Smilde & Geladi (2004).

Fig. 6. Raman data matrix: A graphical representation of the data set obtained from NS
(i )
Raman experiments, where i is the sample number, IRaman (ν̃k ) is the Raman intensity at the
k’th energy position in the Raman spectrum.

Assuming that the Raman data from an experiment involving several samples are collected in
a matrix denoted X, where the matrix element xik represents the measured Raman intensity
for the i th sample at the energy ν̃k in the Raman spectrum. The number of samples are termed
NS , where i = 1, 2, ...., NS and each Raman spectrum consists of NR data points, where k =
1, 2, ...., NR . The data set, containing the Raman intensities for all samples, will then represent
a 2-way multivariate data set, which has the dimensions NS × NR . A graphical representation
of the above is shown in figure 6.
Another way to represent these data is described as follows. The NR data points define
a NR dimensional coordinate system, where the axes are defined by the energies ν̃k . The
Raman spectrum for the i’th sample is then represented by a single point, where the position
of the point is determined by the Raman intensities at the different energies of the Raman
spectrum. The distribution of the NS sample points reflects the systematic differences between
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the Raman spectra of the individual samples. This means that sample points, which represent
samples with similar Raman spectra, will lie close together. In general the dimension NR of
this coordinate system is large and some of these dimensions account for similarities in the
Raman spectra, i.e. no Raman signal (noise) or identical features in the spectra.
The overall principle behind PCA is to make a favorable coordinate transformation, which
represents the significant intensity variations in the spectra. The transformation is in principle
carried out by defining a new rotated coordinate system of dimension NC , where NC is
the number of principal components and typically it is found that NC  NR . For that, a
step-by-step procedure is applied. If we assume that the same spectroscopic structure (not
necessarily with the same absolute intensity) is present in the Raman spectra in the majority
of the samples, then the distribution of the sample points define an average direction in the
NR coordinate system. The unit vector defining this direction is termed the loading vector
p1 , which is calculated from the data by least squares minimization. The score, ti1 , for the i th
sample point, is defined as the coordinate of this point in the direction of the loading vector, p1 .
The first principal component is calculated as the outer product (denoted ⊗) between the score
and the loading vector, and hence is a matrix. This process is carried out again defining next
a second loading and second score vector, the process is repeated until the desired accuracy
is obtained. In Eq.(2) is given the mathematical expression for the data matrix Hedegaard &
Hassing (2008),
Nc
( NC )
xik = ∑ tic pkc + eik (2)
c =1
(N )
where c is the principal component index and eik C are elements in a matrix, termed the
residual matrix, E ( NC ) , which in the ideal case contains only noise.
The information obtained from the PCA may be visualized in different ways. One may plot the
loadings pkc as a function of the energy index, k, for c = 1,2,3,..., N C . The plot of the c’th loading
is proportional to the average Raman spectrum related to the c’th principal component. If the
Raman spectra for the NS samples are almost identical, the plot of the first loadings will show
an average of the common features of the spectra, whereas the manifestation of the differences
happens in loadings of higher order. Another possibility is to plot the scores, tic , as a function
of the sample index, i for c = 1,2,3,..., N C , which illustrates the contribution to each loading
from each sample. A third option is the score plot, where two of the Nc scores are plotted
against each other. The score plot will show a grouping of the sample points according to how
significant each of the two loadings contribute to the multivariate model. Since the goal of
chemical classification problems is to find the eventual minor differences between the Raman
spectra of the different samples, the two latter type of plots clearly reveal, which of the samples
are classified correctly as well as the uncertainty of the classification.
The coherent properties of the Raman process can be utilized to create a 3-way multivariate
data set. For each sample, the data matrix is constructed by measuring the RADIS data as
depicted in figure 6. The elements of the RADIS data matrix xijk , where the additional index
(i )
j defines the photon energy of the laser, are xijk = IR (ν̃j ν̃k ). The 2-way PCA model, which
was discussed above, may be generalized into a 3-way model, called Tucker 3 A. K. Smilde &
Geladi (2004). A discussion of the mathematical details of applying Tucker 3 on various kind
of spectroscopic data can be found in A. K. Smilde & Geladi (2004). Tucker 3 has proven most
useful, when compared to other 3-way models, for treating Raman based data A. K. Smilde
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& Geladi (2004); Hedegaard & Hassing (2008), which is caused by the high quality of the
"molecular bar codes" produced in both dimensions of the RADIS data matrix. In the Tucker
3 model, the elements of the data matrix can be expressed as:
NA NB Nc
( NA NB NC )
xijk = ∑ ∑ ∑ aia bjb ckc gabc + eijk (3)
a =1 b =1 c =1

(N N N )
where eijk A B C are elements in the residual matrix, aia are elements in the score vector,
b jb , ckc are elements in the loading vectors corresponding to the two dimensions defined
by the excitation energy and the Raman shift, and gabc are elements in a matrix reflecting
the interactions between the various scores and loadings. One "Tucker 3 component",
a a ⊗ bb ⊗cc · gabc , is analogue to one principal component in PCA, tc ⊗ pc .
In the 2-way PCA model, described by equation 2, the scores and loadings are determined in
the 2 dimensions so that the data is represented by fewest possible parameters. In the 3-way
case, the goal is basically the same, but now it is necessary to decompose the three dimensions
simultaneously. It is therefore necessary to calculate two loading vectors describing both the
Raman and the RADIS spectra for each score vector along the sample dimension of the data.
A Tucker 3 model will be applied in subsection 3.2 in the discrimination between molecularly
almost identical constituents, α- and β-carotene, when the amount of one of the constituent is
very small compared to the other.

3. Three real life applications of Raman spectroscopy and multivariate analysis

The applicability of Raman spectroscopy in food analysis is demonstrated through the
discussion of three real life applications, namely 1.) Revelation of a content of minced pork
in minced lamb by applying Raman imaging and multivariate analysis, 2.) Discrimination
between two anti-oxidants in a mixture by applying RADIS and three-way multivariate
analysis and 3.) Non-destructive detection of chlorinated pesticide residues on fruits and
vegetables applying a portable Raman spectrometer and SERS.

3.1 Raman imaging and multivariate analysis in the revelation of a content of pork in
minced lamb
In the first application, it is demonstrated that Raman spectroscopy can be applied to identify
a content of pork in minced lamb products available in e.g. supermarkets. The vision
is to develop a scanning system operated by the consumer, which automatically, fast and
non-destructively controls for a specific content, e.g. pork and reports the outcome to the
consumer. A minced lamb product constitutes an inhomogeneous sample, which consists
of areas of meat and fat. The scanning system should be based on a combination of Raman
measurements and a multivariate analysis, where the laser excitation wavelength has been
chosen in accordance with the transmission spectrum of the film covering the minced meat
tub. The discussion of the application is divided into two parts, part 1. Methodology and
part 2. Implementation. In part 1, the molecular marker(s) in the Raman spectra must be
identified, which enables a distinction between meat and fat. The first part is initiated by
using the Raman imaging system (532 and 632,8 nm excitation) shown in figure 2a to create
a reference Raman data set of isolated fat and meat from pork and lamb. The XY scanning
facility of the Raman imaging system is used in order to automatically create a statistically
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large reference data set. Measurements have been carried out changing the wavelength and
varying the laser power in order to judge, which parameters gave the measurements best
suited for identifying the markers and the multivariate analysis. Since the focal distance is
changed during the measurements a software has been developed in order to maximize the
Raman data by selecting the acceptable Raman spectra from the raw data. The change of
focal distance has to be considered in a practical implementation of the method. The different
measurements have besides been optimized with respect to a correction for the background
signal and normalization. It is found that the spectral region, containing the most information
useable in the identification of molecular markers, is from 500 to 1700 cm−1 . One conclusion
to be drawn from the above measurements is that the wavelength 632,8 nm is more suitable
for further measurements, since the laser wavelength 532 nm is placed in a region of larger
absorption and hence may lead to sample degradation.
Figure 7 shows averaged reference Raman spectra of freshly slaughtered pork and lamb (meat
and fat) obtained with the Raman imaging system, 632,8 nm excitation and the laser power
11 mW on the sample. The numbers given in the brackets are the total numbers of spectra,
which are acquired on different places of a given sample.

Fig. 7. Averaged reference Raman spectra (632,8 nm excitation) from (a.) pork fat (283
spectra), (b.) lamb fat (1493 spectra), (c.) pork meat (48 spectra) and (d.) lamb meat (239
spectra).
The method has been validated by measuring the Raman spectra from samples containing
100% minced pork (meat and fat) and 100% minced lamb (meat and fat). The relative amount
of fat in these samples was not specified, but it was less than 20 percent. The number of
spectra acquired for minced pork is 366 and for minced lamb 1320. The multivariate method
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is based on a Partial Least Squares - Discriminant Analysis (PLS-DA) A. K. Smilde & Geladi
(2004), which, contrary to PCA, is a supervised method, where the data is classified according
to predefined classes, in the present case the four classes: 100% meat and fat from pork and
100% meat and fat from lamb. The application of the PLS-DA is combined with an algorithm,
which is illustrated by the flowchart in figure 8. The results of the validation are shown in
table 1.

Fig. 8. A flowchart illustrating the different steps of the PLS-DA analysis.

Table 1 should be read as follows: The first row in the table corresponds to the first question in
the flowchart and the columns correspond to the questions posed secondly in the flowchart.
It follows from the table that the recognition ratio for 1. fat from lamb is 98.0 %, 2. meat
from lamb is 20 %, 3. fat from pork is 89.0 % and 4. meat from pork 0 %. Notice that no
sample from either lamb or pork has been classified wrongly, i.e. interchanged. The following
percentages of the samples have been classified as unknown: fat from lamb fat 2.0 %, meat
from lamb 80 %, fat from pork 11.0 % and meat from pork 100 %. It follows from the results
that the discrimination between minced pork and lamb can be based on the Raman spectra of
fat alone.
Predicted Content
Sample Content Type Quantity Fat Meat Unknown
1 Lamb 1257 724 10 523
100% Minced Lamb Pig 0 0 0 0
Unknown63 15 40 0
2 Lamb 0 0 0 0
100% Minced Pig Pig 313 299 0 14
Unknown53 37 13 3

Table 1. In the table are listed the results from a programmed algorithm for discrimination
between minced lamb (1320 spectra) and pork (366 spectra). The Raman spectra from
different points on the two kind of samples are obtained by an automatized XY scanning and
632.8 nm excitation (11 mW) using the Raman imaging system shown in figure 2a.
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Further information about the experiments and multivariate analysis can be obtained from
the authors ([email protected]).
2. Implementation. A practical system based on a commercial, portable Raman instrument
using 785 nm is under development. The implementation requires that one must consider
the following: a.) localization of the areas of fat by using a machine vision system, b.)
automatized scanning in order to obtain Raman spectra from fat, c.) automatic adjustment
of focus d.) optimization of the excitation volume and laser power, e.) optimization of the
statistics (the scanning area, the relative fat content in minced lamb and pork, respectively and
the determination of the lower limit with respect to revealing a content of pork in a minced
lamb product).
A further development will include the examination of other types of meat than pork and
lamb, investigation of samples subjected to different pre-treatments such as freezing or storing
inside or outside of a refrigerator. Research on these matters are in progress by the authors
of the present paper and in reference Herrero (n.d.). The monitoring of meat quality with
respect to molecular composition as a result of decomposition over time (ageing) applying
a non-invasive and mobile system based on Raman spectroscopy and fluorescence has been
performed in reference G. Jordan (n.d.). Besides, a micro-system including micro-optics and
a compact external laser diode cavity with emission wavelength 671 nm suitable for Raman
spectroscopy has been developed. The overall dimensions of the micro-system light source is
13×4×1 mm3 .

3.2 Discrimination between two nearly identical anti-oxidants by applying RADIS and a
Tucker 3 multivariate model
Carotenoids are a large family of pigments divided into two main groups carotene and
xanthophyll, over 600 different pigments exist. Many of the carotenoid pigments are
ubiquitous in nature and has attracted great interest in health and food science due to their
nutritional importance Coultate (2002); H. D. Belitz & Schieberle (2004); K. Davies (2004). This
importance also most importantly covers the composition of carotenoids in food products
rather than specific individual pigments, this topic is subject to an ongoing discussion. This
highlights the importance of being able to distinguish between very closely related molecular
species, such as lutein, α- and β-carotene.
The second application is a chemical classification problem, in which one must discriminate
between pure β-carotene and a mixture of α- and β-carotene. The challenge is that α- and
β-carotene have nearly identical Raman spectra.
In reference H. Schulz & Baranski (2005), NIR-FT Raman spectroscopy has been applied
in-situ in the analysis of intact plant material, in which the carotenoids are present in their
natural concentrations. The carotenoids are present in a large variety of vegetables and
fruits: orange, carrot roots, red tomato fruits, green French bean pods, broccoli inflorescence,
orange pumpkin, corn and red pepper as well as nectarine, apricot, and watermelon. The
spatial distribution of some carotenoids has been obtained by 2D Raman imaging. Although,
the Raman spectra and images were recorded with a research, laboratory instrument (with
1064 nm excitation and spectral resolution 4 cm−1 ), the same measurements could probably
also be obtained with one of the recently commercially available portable FT-IR instruments
combined with XY translational stage. The analysis performed in reference H. Schulz &
Baranski (2005) is based on the three most intense Raman bands of the carotenoids around
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1500 (ν1 band), 1150 (ν2 band) and 1000 (ν3 band) cm−1 , also shown in figure 1. These bands
are characteristic for all carotenoids, but depending on the specific carotenoid molecule, small
vibrational shifts of the mentioned bands will be observed. The Raman spectra of the pure
carotenoid standards, β-carotene, α-carotene and lutein can be found in figure 3 in reference
H. Schulz & Baranski (2005).
In the analysis performed in H. Schulz & Baranski (2005) regarding anti-oxidants in tomatoes,
the presence of the 1510 cm−1 Raman band has been interpreted as lycopen. A closer
examination of the 1510 cm−1 band shows a small a-symmetry, which may indicate that
β-carotene, where ν1 = 1515 cm−1 , is also present but in a smaller concentration. Additional
studies verify this interpretation. If the molecular species that contribute to the spectra are
more similar than β-carotene and lycopen, the shifts of the corresponding Raman bands
would be smaller and comparable to or less than the resolution of a portable spectrometer.
Furthermore, if the difference in carotenoid concentrations is larger, it may be difficult or even
impossible to solve a classification problem based exclusively on the vibrational shifts. The
spectral distribution in the visible absorption spectra of most carotenoids are rather similar,
however small variations in their color are observed. The change in color reflects small
differences of the energy of the excited electronic states.
In the following we demonstrate how a small shift in electronic absorption energy can be
utilized in a multivariate analysis of the vibrational Raman data. We consider a classification
problem, in which the goal is to discriminate between samples with pure β-carotene and
samples with a mixture of α- and β-carotene, where the concentration of α-carotene is very
small compared to the concentration of β-carotene. From the Raman spectra of the carotenoid
standards, it follows that α- and β-carotene have nearly identical Raman spectra: 1515 cm−1
(1521), 1156 cm−1 (1157) and 1007 cm−1 (1006), where the numbers in the brackets correspond
to α-carotene.
The classification is performed by applying a Tucker 3 multivariate analysis to the RADIS data
matrix obtained with the laser wavelengths 476.5, 488 and 497 nm. When comparing the laser
wavelengths with the maxima of the absorption spectra of α- and β-carotene in references
(n.d.a); H. D. Belitz & Schieberle (2004); Miller (1934), it is seen that we excite the carotene
molecules close to resonance with the lowest electronic transition. The shift in electronic
energy can be estimated from the absorption spectra to 268 cm−1 . In the RADIS spectra, this
shift will manifest itself as a different resonance enhancement of the Raman signal of the two pigments
for each laser wavelength. The RADIS data matrix will therefore from sample to sample vary
along the two dimensions defined by the excitation energy and the Raman shift.
The experimental Raman and RADIS data are obtained from 10 samples, where 5 samples (1 -
5) contain only β-carotene and 5 samples (6 - 10) contain a mixture of 10% α-carotene and 90%
β-carotene. The Raman spectra from one sample (476.5 nm excitation) of β-carotene (dashed
line) and the mixture of α-carotene and β-carotene (solid line) in solution are shown in figure
9.
The results of a PCA and Tucker 3 analysis of the data are shown in figure 10.
Details about performing the Tucker analysis on similar RADIS data are given in Hedegaard
& Hassing (2008). In both the PCA and in the Tucker 3 analysis the classification succeeds
and the results are comparable, since the ratios between the average distance between the two
classes and the variation within each class are similar. However, further studies involving
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Fig. 9. The Raman spectra from one sample (476.5 nm excitation) of β-carotene (dashed line)
and the mixture of α-carotene and β-carotene (solid line) in solution.

Fig. 10. (a.) Score plot of a 2 component PCA model, excitation wavelength 476 nm (score 2
(ti2 ) is plotted against score 1 (ti1 ) for i = 1, 2....10), (b.) Score plot of a 4 component Tucker 3
model, 476.5 and 496.5 nm (score 2 (ai2 ) is plotted against score 1 (ai1 )) and (c.) Score plot of a
2 component PCA model, 496.5 nm.

10 samples (1 - 10) with only β-carotene and 10 samples (11 - 20) with a mixture of only
0.5% α-carotene and 99.5% β-carotene show that the PCA in most cases leads to a wrong
classification, whereas the Tucker 3 model in most cases still leads to a correct classification.
The robustness of the PCA and Tucker 3 models with respect to experimental uncertainties,
such as fluctuations in the laser intensity, is different. This is demonstrated in the results
shown in figure 11.
The number of samples and the concentration ratio are the same as in figure 10. In general one
would expect the classification based on a Tucker 3 analysis to improve by incorporating an
extra wavelength. However, by comparing figure 11b with 10b, it is seen that the classification
succeeds, but the spread within the classes has increased. The result of the PCA analysis in
11a shows that the PCA classification fails, since it leads to a false grouping of the samples. A
closer study of the experimental conditions reveals that the laser intensity at 488 nm fluctuates,
which is the explanation for the above results.
In many cases the PCA or PLS-DA analysis of Raman data works fine due to the well resolved
spectra. However, it is, as demonstrated above, possible to improve the analysis by including
a shift in electronic absorption energy. Another possibility is to include the polarization
properties of the Raman spectra in the analysis S. Hassing (2011).
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Fig. 11. (a.) Score plot of a 2 component PCA model, excitation wavelength 488 nm (score 2
(ti2 ) is plotted against score 1 (ti1 ) for i = 1, 2....10), (b.) Score plot of a 4 component Tucker 3
model, 476.5, 488 and 496.5 nm (score 2 (ai2 ) is plotted against score 1 (ai1 ) for i = 1, 2....10).

3.3 Non-destructive detection of chlorinated pesticide residues on fruits and vegetables

applying a portable Raman spectrometer and SERS
We are today facing an increasing exposure to a cocktail of pesticides and other chemicals.
The daily exposure to these chemicals, often used in horticulture, agriculture etc., is suspected
to cause issues in human health, these include several diseases, reduced fertility and birth
defects. One contribution to this exposure is the daily intake of pesticides through food
consumption. Besides, an increasing concern with respect to the "cocktail effect", arising when
digesting food products containing several pesticides, is seen, since the joint action is not
fully understood Danish Ministry of the Environment (2005). Today, more than 800 different
pesticides are available and a large number of those are applied to food products Danish
Environmental Protection Agency (2005); Veterinary et al. (2003; 2005). Gas Chromatography
(GC-multi method, FP017) has been the method of choice for such determination, this
procedure, however, is both labor intensive, destructive and non-portable Veterinary &
Administration (2003). A calculated intake of pesticides is found by multiplying the average
consumption of a commodity with the content of the pesticide and a Hazardous Quotient
(HQ) is found by dividing the above mentioned intake with the Acceptable Daily Intake (ADI).
The most frequently found pesticides were not including those contributing the most to the
hazard quotient, but e.g. dicofol.
It should be noticed that although dicofol is toxic, building up sediments in plants and animals
and therefore banned in e.g. Denmark, it has in a spot check been detected on fruit and
vegetables imported to Denmark Veterinary et al. (2003). The detection of the pesticide may
be due to either contaminated soil, due to a long decomposition time, or due to legal/illegal
use in some countries. The acaricide has commonly been applied to a number of fruits among
others oranges, apples, grapefruit, lemon, mandarin, clementine, pears, table grape, exotic
fruit and tomatoes Veterinary et al. (2003).
The goal of the third application to be discussed is hence a non-destructive, in-situ revelation
of the banned pesticide, the organochlorine acaricide and insecticide dicofol, on tomato, apple
and carrot by using a portable Raman spectrometer and SERS. A portable system will enable
initial spot checking, e.g. in harbors or supermarkets.
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As already mentioned, it is not unproblematic to apply SERS or SE(R)RS, since the molecular
species should be in close proximity to a nano-structured metal surface in order to obtain an
enhanced Raman signal and since the SERS or SE(R)RS spectra (the "molecular bar code")
may be different from the Raman spectra of the free molecule. The Raman spectra can for
some pesticides in a solution even with a concentration much higher than the detection
limit not even be measured. If more pesticides are anticipated on a commodity, the more
molecular information available and the more reliably the detection of even structurally
related pesticides should be done. Besides, a quantitative analysis based on SERS/SE(R)RS
requires in general special attention. Some of the approaches that can be used to ensure that
the pesticide is brought reproducibly close to the surface are 1. the pesticide is attached to the
molecular species functionalized to the surface of either the colloid or the substrate or 2. the
molecules used to functionalize the surface provides molecular pockets, where the pesticide
can be trapped close to the surface L. Guerrini (2008). The functionalization, may complicate
the SERS spectra, which could necessitate the application of multivariate analysis to elucidate
the SERS response from the molecular species under investigation (molecular bar code blurred
due to interference from functionalization molecules).
The SERS spectra of an organochlorine pesticide deposited on an Ag substrate, similar to
the one shown in figure 12, are earlier reported without any measures taken to ensure
the molecular position close to the surface Alak & Vo-Dinh (n.d.). Applying the pesticide
reproducibly to the surface itself is an act of art, especially when the foundation for a
reproducible, quantitative measurement is required. This touches upon the question of how
well the concentration is known, when the pesticide has been applied to the surface. In
reference J. C. S. Costa & Corio (n.d.) the behavior of Au nano rods and Ag nano cubes as high
performance SERS sensors has been evaluated for amongst others a chlorinated pesticide in
a 10−7 M solution. In J. C. S. Costa & Corio (n.d.); L. Guerrini (2008) the SERS spectra were
obtained by using research instruments.
In the present study a silver Film Over Nano spheres (AgFON) is used as the surface
enhancing substrate and the portable Raman spectrometer (DeltaNu InspectorRaman shown
in figure 2) as ’the analyzing unit’. The AgFON and periodic particle array (PPA) substrates
fabrication process is shown in figure 12. This latter type of substrate can be fabricated
according to the same principles governing the fabrication of AgFON’s, the differences are,
that the PPA’s must be made on a transparent material, i.e. cover glass, and the final step
is a lift-off of the nano spheres and the metal covering these, leaving only the triangular
shaped deposits on the glass. Details about the fabrication process can be found in references
A. Henry & Duyne (n.d.); Willets & Duyne (n.d.).
Conventional Raman spectra from the mentioned commodities were first acquired in order to
be able to judge the the possible intervenience from the background spectra, see figure 13.
The normal Raman spectrum of the pure pesticide (figure 14) is initially recorded and
compared to the pesticide reference spectrum obtained in reference M. L. Nicholas & Bromund
(1976) and the obtained SERS spectra as well. In this way it is possible to detect a pesticide,
which was not detected by using conventional Raman spectroscopy and to identify the
individual peaks from the pesticide in the SERS spectrum.
Next, in figure 15 are seen a Raman spectrum of a 10−3 M pesticide dissolved in methanol
and a SERS spectrum of the same solution as well as the spectrum of the bare nanostructured
surface. The substrate of the type AgFON is seen in the figure as well.
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Fig. 12. A schematic representation of the fabrication procedure, when making AgFON’s or
PPA’s using thermal vapor deposition (A. Henry & Duyne (n.d.); Willets & Duyne (n.d.)). In
the case of the AgFON, the wafer used in the present research has been copper and the wafer
used in the PPA is cover glasses. The metal used is Ag and the thickness of the layer 200 nm
and the spheres used are polystyrene spheres.

Fig. 13. Conventional Raman spectra of tomato, apple and carrot acquired with Inspector
Raman.

The Raman spectrum of the pesticide solution reveals no presence of dicofol only the Raman
spectrum of the solvent is observed underlining the need for enhancement. In the SERS
spectrum the peak at 1096 cm−1 indicates the presence of dicofol, which is seen by comparing
the spectrum of dicofol powder with the SERS spectrum. The SERS spectrum contain several
Raman bands, but by comparing the spectrum of the bare substrate with the SERS spectrum,
it is seen that these overlap with Raman bands found on the bare AgFON. This stresses the
need for developing a handling/cleaning procedure or a protective layer for the substrates.
In the implementation of an on-site detection of pesticides on fruits and vegetables, the
idea is that the substrate must be transmission based in stead of reflection based as used
presently. This will possibly enable a closer contact with the fruit or vegetable surface and a
penetration of the laser through the PPA to the commodity, the scattered Raman signal should
be detected through the thin glass surface as well. Since dicofol, does not form covalent
bonds with the surface, but interacts via other more weak forces, it is difficult to obtain
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Fig. 14. A Raman spectrum of a pure dicofol powder, which was acquired with Inspector
Raman. Notice the peak at 1099 cm−1 .

Fig. 15. Raman spectrum of a 10−3 M pesticide solution (blue), the SERS spectrum of the
same solution (red) and a Raman spectrum of the bare AgFON. The solvent is methanol.

reliable and quantitative measurements without any measures taken to ensure a reproducible
binding to the surface. A functionalization with albumin, exploiting the high lipid affinity
of organochlorine pesticides, and/or albumin affinity, may be the answer to this problem
M. Gülden & Seibert (2002); Maliwal & Guthrie (1981); Moss & Hathway (1964). Besides, a
complete displacement of the surface contamination does not take place, which may cause
a large background signal. The toxic nature of the pesticides complicates the optimization
process of the SERS measurements on these.
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In reference (n.d.b); C. Shende & S. Farquaharson (n.d.) it is demonstrated that single

pesticides (phosphor- and nitrogen containing) and a mixture of pesticides can be detected
fast (5 minutes) in low concentrations (50 - 100 ppm). The SERS setup included a portable
FT-Raman spectrometer (785 nm) and specially prepared SERS active capillary probes for
chemical extraction of the pesticides and generation of the SERS signal.
Further information about the experiments and fabrication of the substrates as well the
possibilities for implementation can be obtained from the authors ([email protected]).

4. Outlook
Raman spectroscopy has through many years been recognized as an advanced research
tool for obtaining detailed molecular information. Depending on how the experiments
are preformed different kind of information can be revealed such as molecular structure,
dynamics and functions of bio-molecules . All due to the coherent nature of the Raman
scattering process. Raman spectroscopy meets almost all the requirements listed in the
introduction and has, due to the development of highly sensitive CCD-detectors, a variety
of small laser sources, hand held spectrometers (dispersive and FT-instruments)and fast
computers for the implementation of advanced data processing, the potential for being a fast,
non-destructive and molecule specific tool for inspection of food quality. Despite this, Raman
spectroscopy has not yet become a standard method within control of food quality, especially
not as a standard on-site inspection technique. Our aim has not been to write an exhaustive
review article about application of Raman scattering in food analysis Li-Chan (1996), but
merely to present an adequate and pinpointed amount of theory and experimental aspects
through which an increased understanding enables a more inspiring, creative and intelligent
access to applying some kind of Raman spectroscopy within food applications. One major
challenge is still to transform the Raman technique from research and analytical laboratories
into real life applications, especially the detection of trace amounts of unknown, unwanted
substances may be a challenge. We have demonstrated, through the discussion of three rather
different case studies, that it is possible, but it requires additional developing work to be
performed. Before one attempts to meet the practical challenges, it is therefore mandatory to
understand the practical hurdles to be overcome in order to benefit from the high molecular
selectivity that Raman spectroscopy can offer. Although there are remaining problems to be
solved, we believe that within a few years Raman spectroscopy will develop into the area of
analyzing food quality, on-site and non-destructively.

5. References
(n.d.a).
(n.d.b).
A. Henry, J. M. Bingham, E. R. L. D. M. G. C. S. & Duyne, R. P. V. (n.d.). J. Phys. Chem. C .
A. K. Smilde, R. B. & Geladi, P. (2004). Multi-way Analysis: Applications in the Chemical Sciences.
Alak, A. M. & Vo-Dinh, T. (n.d.). Analytica Chimica Acta .
C. Shende, F. Inscore, A. G. P. M. & S. Farquaharson, Journal = Nondestructive Sensing for Food
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 5

Contamination of Foods by Migration

of Some Elements from Plastics Packaging
O. Al-Dayel, O. Al-Horayess, J. Hefni, A. Al-Durahim and T. Alajyan
King Abdulaziz City for Science and Technology, Riyadh,
Saudi Arabia

1. Introduction
There are various types of packaging material including paper, board, plastic, metal, glass,
wood and other materials. Paper and board packaging accounted for the largest share of
global packaging sales in 2003 with 39% of the total. Plastic packaging accounted for 30%
(rigid and flexible plastics) of the market, with metal packaging accounting for 18% and
glass packaging a further 7%. Other packaging products accounted for the remaining 6% of
the market. Rigid plastics was the fastest growing sector of the market during the period
1999-2003. Around 70% of overall consumer packaging consumption is used for food and
beverage packaging (WPO, 2008). Many different types of plastics are being used as
packaging materials. The key components in plastic materials are polymers which are made
of units of organic material, and one or more of large molecular weight can be formed as
desired. Most polymers are petrochemical compounds with additive materials to give them
properties of flexibility, elasticity and resistance to fracture and transparency to light ( Oi-
Wah & Siu-Kay, 2000; Al-Dayel et. al. 2009)
The final plastic material thus is a mix of polymer, additives, manufacturing aids, and side
products from the complex polymerization process that were not intentionally added
(Bradley & Coulier 2007).
Low density Polyethylene (LDPE) is used in preparation of most of the hot food packaging.
LDPE has high flexibility, and can be affected by organic solvents. It has melting
temperature of 110 ° C (wikipedia.org 2012).
High Density Polyethylene (HDPE), has the same uses as those of the low density, but it is
much flexible and resistant to organic solvents and to high temperatures. It is used in
manufacture of some household appliances, pipes and hoses. It is also used in food
packaging which are subject to sterilization temperatures. HDPE is characterized by its
ability to isolate the humidity, and its flexibility even at freezing temperature.
Different types of additives, such as antioxidants, stabilizers, lubricants, anti-static and anti-
blocking agents, have been developed to improve the performance of polymeric packaging
materials (Achilias 2007; Susan1992).
The role of food and beverage packaging as a source of contaminants have raised many
concerns after the widespread use of such containers; packaging. Any substance (monomers
74 Food Quality

and other starting substances, additives, residues) which migrates from the packaging into
the food is of concern if it could be harmful to the health (Donatella et. al., 2010; Grob et. al.,
2006).
The migration of additives or contaminants from polymeric food packaging to food may be
separated into three different, but inter-related, stages: diffusion within the polymer,
solvation at the polymer food interface, and dispersion into bulk food (Oi-Wah & Siu-Kay,
2000). The migration has been showed to increase with fat content and storage temperature
(Sanches, et al.,2007).
Antimony does not bioaccumulation, so exposure to naturally occurring antimony through
food is very low. Antimony is present in food, including vegetables grown on antimony-
contaminated soils, mostly in the low μg/kg wet weight range or less (WHO, 2003).

Antimony toxicity is dependent on the exposure dose, duration, route (breathing, eating,
drinking, or skin contact), other chemical exposures, age, sex, nutritional status, family
traits, life style, and state of health (Ross and Adrian, 2009). Chronic exposure to antimony
in the air at levels of 9 mg/m3 may exacerbate irritation of the eyes, skin, and lungs (Roper,
1992). Long-term inhalation of antimony can potentiate pneumoconiosis, altered
electrocardiograms, stomach pain, diarrhea, vomiting, and stomach ulcers, results which
were confirmed in laboratory animals (Roper, 1992). Although there were investigations of
the effect of antimony in sudden infant death syndrome, current findings suggest no link.
Long-term exposure in experimental animals has shown an increase in the hepatic
malfunction and blood changes (ATSDR, 1992). It is not clear yet whether antimony is a
human carcinogen. Occupational epidemiology could not confirm evidence of lung
carcinogenicity caused by antimony as detected in female rats (Gerhardsson et al. 1982;
Jones, 1994; Groth et. al.1986). Furthermore, because the experimental results were not
uniform, animal lung carcinogenicity by antimony is still a matter for debate (Jones, 1994;
Newton et. Al., 1994; Ross and Adrian, 2009).
This work is an attempt to evaluate the migration of elements from packaging materials into
food stuffs. A severe contact condition between food and packaging materials has been
created to evaluate the maximum potential migration of elements (Grob et. al.1999).

2. Experimental
2.1 Samples
Two types of polyethylene samples have been selected from the Saudi market. One is called
thermal bag which is commonly used for hot food, and the other one is called food bag
which is commonly used for cold and freezing food. Both of these types are made of liner
low density polyethylene. The samples elemental concentrations have been obtained using
neutron activation analysis (NAA).

2.2 Neutron Activation Analysis (NAA)

Samples were irradiated at the McMaster University Reactor in Hamilton, Ontario, Canada.
It is a 2MW open pool research reactor.
Contamination of Foods by Migration of Some Elements from Plastics Packaging 75

The Gamma ray spectrum acquisition was carried out by the use of a high resolution
intrinsic germanium detector.
Two portions of each sample were weighed into a plastic irradiation container. One portion
is for short-lived isotope analysis (half life < 24 hours). This portion is about 4 grams
weighed into a 7 ml volume container, and it was irradiated for about 60 seconds using a
thermal neutron flux of approximately 6x1012 n/ cm2.s . After a short decay period of 6
minutes, the gamma ray spectrum was acquired. The sample was then allowed to decay for
further period of 24 hours.
The other portion was for longer lived isotope analysis (half life >5 days). Approximately 24
grams of the sample was weighed into a 40 ml container, and was irradiated for 20 minutes
using thermal neutron flux of approximately 8x1012 n/ cm2.s. After 3 days of decay, the sample
was counted for 60 minutes. The sample was again allowed to decay for further period of 18
days and then counted for 2 hours. Results were calculated for each of the four spectra.

2.3 Quality assurance for NAA analysis

To assess the analytical process and make a comparative analysis, Standard Reference
Material coal sample (SARM-18) from South African Bureau of Standards and a trace
element in coal sample from the USA National Institute of Standards and Technology
(NIST) (SRM 1632c) were analyzed in the same manner as other samples (Anderson &
Cunningham, 2000; Wang & Sakanishi, 2004). Table (1) gives the comparison of the certified
values and these obtained in this work for each reference materials. The results are generally
in a good agreement except for Sr, Zr, Ba, Sb, Ba, Ca, Eu, Na, and As in one or other
standard reference material samples.

Element in SARM 18 NIST 1632c

mg/kg
(%) This work RSD Range@ This work RSD Certified Value
(µg/Kg)

Al (%) 1.34 3 2.54-2.61 0.94 4 0.915±0.0137

Sb 0.30 5 0.3* 0.22 5 0.461±0.029
As 0.52 4 3.5 3 6.18±0.27
Ba 91 6 71-82 70 5 41.1±1.6
Br 3.5 3 3* 20.9 3 18.7±0.4
Cd 0.7 20 <0.3 0.072±0.007
Ca (%) 0.138 4 0.17-0.19 0.21 4 0.145±0.03
Ce 20.6 3 21-24 8.2 3 11.9±0.2
Cs 1.17 4 1* 0.473 5 0.594±0.010
Cl 54 5 1130 3
Cr 17.3 3 14-18 10.4 3 13.73±0.20
Co 7.5 3 5.5-7.2 3.4 3 3.48±0.2
Cu 4.9 9 6.7 6 6.01±0.25
Dy 1.87 3 0.57 3
76 Food Quality

Eu 0.31 3 0.3* 0.17 3 0.124±0.003

Ga 7.1 5 8* 2.6 6 3*
Au 0.65 23 <0.5
(µg/Kg)
Hf 1.9 3 1.7-1.9 0.44 4 0.585±0.010
In 0.028 12 0.02 11
I 1.0 11 1.5 6
Ir (µg/Kg) <0.7 <0.5
Fe (%) 0.22 3 0.28-0.29 0.77 3 0.735±0.011
La 10.6 3 9.0-13 4.7 3
Lu 0.207 3 0.055 4
Mg (%) 0.056 6.5 0.1-0.11 0.039 8 0.0384±0.0032
Mn 21.5 3.2 21-23 13.5 3 13.04±0.53
Hg <0.017 0.04* 0.0802 8 0.0938±0.0037
Mo 1.13 5 0.693 9 0.8*
Nd 7.5 5 3.1 5
Ni <10 <6.8 9.32±0.51
K (%) 0.12 4 0.140-0.150 0.081 3 0.11±0.0041
Rb 8.3 6 6.7-9.5 5.4 6 7.52±0.33
Sm 1.7 5 1.9-2.2 0.72 5 1.078±0.028
Sc 5.1 3 4.0-4.7 2.0 3 2.905±0.036
Se 0.2 31 1.1 5 1.326±0.071
Ag <0.2 <0.1
Na (%) 0.013 3 0.052 3 0.03±0.005
Sr 31 15 42-45 109 4 63.8±1.4
Ta 0.36 3 0.3* 0.14 3
Te <0.9 <0.3 0.05*
Tb 0.29 6 0.3* 0.10 9
Th 3.8 3 3.0-4.3 1.2 3
Sn <11 1* <7 1*
Ti 0.067 3 0.111-0.116 0.047 4 0.052±0.003
W 1.5 3 2* 0.4 6
U 1.9 3 1.5-2.0 0.40 4 0.513±0.012
V 22 3 21-25 15 4 23.72±0.51
Yb 1.26 3 0.34 4
Zn 6 10 10 6 12.1±1.3
Zr 20 19 62-71 <15 16*

@ Range refers to 95% confidence limits.

* Uncertified value.
RSD: Relative Standard Deviation.
Table 1. Comparison of elemental concentration of the standard reference material (SARM18
and NIST 1632c) in this work and their certified values.
Contamination of Foods by Migration of Some Elements from Plastics Packaging 77

2.4 Study of elemental migration

Four types of food stuffs were examined, water, 30% ethanol, olive oil and 5% acetic acid.
The aim was to study the immigration of Al, Sb, Cu, Mg, Ti and Zn.
To assess the maximum possible value of elements migration, the samples were prepared
under the influence of severe contact conditions between the bag materials and the food stuff.
This influence have been created by mixing the bag material, in the form of powder, with the
food stuffs and exposing the mixture to high temperature (80-100 °C). Such a contact case
cannot be exist in normal human use, but it can show the maximum potential migration.

2.5 Experimental arrangement and ICP-MS analysis

Four samples were prepared in four flasks. Each flask contained a mix of 2.5 g of the sample
powder and 25 ml of one of the four food stuffs. The flasks were placed in a shaker at 160
rpm stirring rate at temperature of 100 oC (80 oC for ethanol case).
The samples were elementally analyzed using a Perkin-Elmer Sciex Instruments multi-
element ICP-MS spectrometer, type ELAN6100, equipped with a standard torch, cross flow
nebulizer and Ni sampler and skimmer cones.

2.6 Quality assurance for ICP-MS analysis

To assess of the analytical process and make a comparative analysis, Standard Reference
Material (SRM), Nist-1640 Natural Water purchased from the National Institute of
Standards and Technology (NIST), USA was analyzed in the same manner as all other
samples. Table 2 compare the certified values with those obtained in this work. The results
are generally in good agreement with the certified values.

3. Results and discussion

The elemental concentrations in the samples and the corresponding concentrations migrated
to food stuffs are shown in table 3. The results show that the Antimony (Sb) has the highest
migrated concentration ratio (37 %), in the acetic acid, followed by Zink (Zn) with 22 % ,
Magnesium with 17 %, in ethanol and Titanium (Ti) with 12%, in olive oil. The lowest
migration ratio was that of Aluminum (Al) which was only 0.012 %, in ethanol. Table 3:
Concentration of elements in bags materials and migrated to food stuffs.

4. Conclusion
The NAA and the ICP-MS analytical methods used in this work gives good results. These
results were confirmed by the analysis of the standard reference materials as shown in
tables 1 and 2.
Migration of substances from plastic packaging materials into food stuffs is clearly
measured in the conditions which have been created to represent the worst case of contact
between packaging material and food stuff. This suggests that a further studies on migration
of substances from plastic packaging into food stuffs in the normal conditions should tack
place. The results also suggest expected a seriousness harmful effect of using wrong plastic
packaging material for heating foods in microwave ovens.
78 Food Quality

Elements This Work Certified values

Concentration in RSD Concentration in RSD
ppb ppb
Li 49.8 1.50 50.7 2.76
Be 36.4 4.95 34.94 1.17
B 264 8.60 301.1 2.03
Na 27800 0.34 29530 1.05
Mg 5800 0.50 5819 0.96
Al 47.4 0.32 52 2.88
K 899 0.93 994 2.72
Ca 6990 0.97 7045 1.26
V 11.9 1.16 12.99 2.85
Cr 37.1 0.45 38.6 4.15
Mn 117 0.46 121.5 0.91
Fe 35.2 15.65 34.3 4.66
Co 19 0.50 20.28 1.53
Ni 27 1.19 27.4 2.92
Cu 89.2 0.19 85.2 1.41
Zn 64.7 1.36 53.2 2.08
Ga 0.198 30.20
As 27.3 1.59 26.67 1.54
Se 23.7 3.95 21.96 2.32
Rb 2.22 1.65 2 1
Sr 108 0.40 124.2 0.56
Mo 43.5 2.04 46.75 0.56
Ag 6.48 0.39 7.62 3.28
Cd 21.9 2.14 22.79 4.21
Sb 12.6 2.85 13.79 1.46
Te 0.328 26.89
Ba 139 0.96 148 1.48
Tl 0.146 42.60 <0.1*
Pb 26.8 0.79 27.89 0.50
Bi 0.143 40.49
U 0.834 7.41
RSD: Relative Standard Deviation.
Table 2. Comparison of elemental concentration of the reference material ( NIST 1640) in this
work with the certified values.
Contamination of Foods by Migration of Some Elements from Plastics Packaging 79

Table 3. Concentration of elements in bags materials and migrated to food stuffs.

5. References
Achilias D. S., Roupakias C., Megalokonomosa P., Lappas A.A., Antonakou E.V. (2007 )
Chemical recycling of plastic wastes made from polyethylene (LDPE and HDPE)
and polypropylene (PP) ElsevierB.V. (available online)
Agency for Toxic Substances and Disease Registry (ATSDR), (1992) Toxicological profile for
antimony. Atlanta, Georgia, USA: US Department of Health and Human Services,
Public Health Service; 1992. p. 160
Al-Dayel O., Al-Horayess O., Hefni J. and Al-Durahim A.,(2009) Trace Elements in
Packaging Polymers, Research Journal of Chemistry and Environment, 13, 1, 92
Anderson D and Cunningham W, (2000) Revalidation and Long-Term Standard Reference
Materials, J.AOAC.Int.,83, 5, 1121
Bradley, E and Coulier, L, (2007) An investigation into the reaction and breakdown products
from starting substances used to produce food contact plastics, Central Science
Laboratory, London.
Donatella Restuccia , U. Gianfranco Spizzirri , Ortensia I. Parisi, Giuseppe Cirillo, Manuela
Curcio, Francesca Iemma, Francesco Puoci, Giuliana Vinci, Nevio Picci, (2010) New
EU regulation aspects and global market of active and intelligent packaging for
food industry applications, Food Control 21, 1425
Gerhardsson L, Brune D, Nordberg G.F, Wester P.O, (1982) Antimony in lung, liver and kidney
tissue from deceased smelter workers, Scand. J. Work Environ. Health, 8 , pp. 201–208
Grob K, Spinner C, Brunner M, Etter R. (1999) The migration from the internal coatings of
food cans; summary of the findings and call for more effective regulation of
polymers in contact with foods: a review. Food Addit Contam;16. 579
Grob K, Biedermann M, Scherbaum E, Roth M, Rieger K. (2006) Food contamination with
organic materials in perspective: packaging materials as the largest and least
controlled source? A view focusing on the European situation. Crit Rev Food Sci
Nutr;46, 529
80 Food Quality

Groth D. H, Stettler L.E , Burg J.R, Busey W.M, Grant G.C, Wong L,(1986) Carcinogenic effects
of antimony trioxide and antimony ore concentrate in rats, J. Toxicol. Environ. Health,
18 ), pp. 607–626
Jones R.D , (1994) Survey of antimony workers: mortality 1961–1992, Occup. Environ. Med.,
51 , pp. 772–776
Newton P.E., Bolte H.F., Daly I.W., Pillsbury B.D., Terrill J.B., Drew R.T., Ben Dyke R,
Sheldon A.W., Rubin L.F.,(1994) Subchronic and chronic inhalation toxicity of antimony
trioxide in the rat, Fundam. Appl. Toxicol., 22, 561–576
Oi-Wah Lau , Siu-Kay Wong (2000) , Contamination in food from packaging material,
Journal of Chromatography A, 882, 255
Roper WL, editor. (1992) Toxicological Profile for Antimony and Compounds. Atlanta, Georgia,
USA: Agency for Toxic Substances and Disease Registry. Public Health Statement;
pp. 1–5
Ross G. Cooper and Adrian P. Harrison, (2009 April) The exposure to and health effects of
antimony, Indian J Occup Environ Med. 13(1): 3–10
Sanches Silva, A, et al.,(2007) Kinetic migration studies from packaging films into meat
products, Meat Science 77, 238
Susan Brewer M. (1992). , Cooperative Extension Service, University of Illinois at Urbana-
Champaign, Circular 1320A.
Wang J, Nakazato T and Sakanishi K, (2004) Microwave digestion with HNO3/H2O2
mixture at high temperatures for determination of trace elements in coal by ICP-
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WHO/SDE/WSH/03.04/74, (2003) Antimony in Drinking-water
WPO (2008) Market Statistics and Future Trends in Global Packaging
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www.wikipedia.org (2012)
 Section 2

Some Case Studies

Improving the Food Quality
 6

Senescence of the Lentinula edodes

Fruiting Body After Harvesting
Yuichi Sakamoto1, Keiko Nakade1,2,
Naotake Konno1 and Toshitsugu Sato1,3
1Iwate Biotechnology Research Center,
2TSUMURA & CO
3Kitami Institute of Technology,

Japan

1. Introduction
Lentinula edodes, or shiitake mushroom as it is more popularly known, is one of the most
economically important edible cultivated mushrooms. However, postharvest spoilage, such
as browning of the gills and softening of the fruiting body, results in loss of freshness and
consequent loss of food value (Minamide et al.; 1980a, b).

Fig. 1. Fruiting bodies a): just after harvest (fresh fruiting body) and b): at 4 days after
harvest (senescent fruiting body).
84 Food Quality

Numerous studies addressing the mechanisms of quality loss during postharvest storage
have revealed that browning of the L. edodes fruiting body is associated with increased
activities of tyrosinase (Tyr; Kanda et al., 1996a, b) and laccase (Lcc; Nagai et al., 2003) after
harvest. In addition to being an important fresh food source, shiitake mushrooms also have
medicinal value. Lentinan, a β-1,3-glucan used for tumor immunotherapy (Chihara et al.,
1969), is purified from fresh shiitake mushrooms. However, lentinan content decreases
during postharvest storage (Minato et al., 1999). The reported structure of lentinan is a β-1,3-
linked-D-glucan with β-1,6 branches (Chihara et al., 1969), and it appears that postharvest
degradation of lentinan during mushroom storage is mediated by β-1,3-glucanase (Minato
et al., 1999). Four glucanases have been reported in L. edodes fruiting bodies, two exo-β-1,3-
glucanases, EXG1 (Sakamoto et al., 2005a) and EXG2 (Sakamoto et al., 2005b), and two endo-
β-1,3-glucanases, TLG1 (Sakamoto et al., 2006) and GLU1 (Sakamoto et al., 2011). An endo-
β-1,6-glucanase, Pus30, was also purified from L. edodes fruiting body (Konno and
Sakamoto, 2011). Except for EXG1, these glucanases are involved in lentinan degradation
after harvesting.
Postharvest changes are considered to be complex, and there is currently little information
about the changes in gene transcription following harvest of the L. edodes fruiting bodies. It
has been reported that the expression of several genes increases during the postharvest
period in Agaricus bisporus (button mushroom, Eastwood, 2001). Eastwood et al., (2001)
revealed that argininosuccinate lyase increases after harvest, and the relationship between
elevated levels of argininosuccinate lyase and postharvest physiological changes of the A.
bisporus fruiting body has been investigated in detail (Wagemaker et al., 2007; Eastwood et
al., 2008). Several studies have analyzed changes in gene transcription during development
of the fruiting body of L. edodes (Hirano et al., 2004; Miyazaki et al., 2005). More recently,
EST analysis (Suizu et al., 2008) and SAGE analysis (Chum et al., 2008, 2011) were carried
out on L. edodes. Changes in gene transcription after harvest of the L. edodes fruiting body
were investigated, revealing that many genes were newly expressed after harvest, such as
putative chitinases, chitosanase, and a transcription factor (Sakamoto et al., 2009). This
chapter will discuss about genes involved in fruiting body senescence in mushrooms,
especially in L. edodes.

2. Phenol oxidases involved in browning of fruiting body after harvesting

The postharvest preservation of L. edodes fruiting bodies causes gill browning, which is
commercially undesirable since it causes an unpleasant appearance. In general, melanins
involved in gill browning are considered to be synthesized from β-(3,4-
dihydroxyphenyl)alanine (DOPA), derived from tyrosine. DOPA can be oxidized
enzymatically to quinones, which polymerize nonenzymatically to form the melanin
pigments. Oxidation of tyrosine is commonly catalyzed by tyrosinasae (Tyr: EC1.14.18.1).
The mechanisms of mushroom browning have been investigated extensively in A. bisporus
(Burton, 1988; Espín et al., 1999). Browning in this species is mainly due to DOPA and c-
glutaminyl-3,4-dihydroxy- benzene (GDHB) melanins (Jolivet et al., 1998), and Tyr seems to
play the most important role in their synthesis (Turner, 1974). Burton (1988) reported that
epidermal tissues of A. bisporus had a greater activity of non-latent Tyr and a greater
concentration of phenols than did the fresh. It has been reported that Tyr activity of L. edodes
fruiting bodies increases after harvest, and a Tyr has been purified (Kanda et al., 1996a).
Senescence of the Lentinula edodes Fruiting Body After Harvesting 85

Laccases (Lccs: EC 1.10.3.2) catalyze the single-electron oxidation of phenols or aromatic

amines to form different products via various pathways. Lcc belongs to a group of
polyphenol oxidases that contain copper atoms in their catalytic center; thus, they are
typically referred to as multicopper oxidases. Lcc catalyzes the single-electron oxidation of
phenolic substrates or aromatic amines to form different products via a variety of
biochemical pathways (Leonowicz et al., 2001). There are many reports in the literature of
the purification and characterization of Lcc isoforms from white-rot fungi, and several Lcc-
encoding genes have been isolated (reviewed by Kumar et al., 2003; Baldrian, 2006). A
correlation between melanin synthesis and intracellular Lcc in Cryptococcus neoformans has
been reported (Ikeda et al., 2002). Lcc in Aspergillus nidulans is considered to form L-DOPA
to synthesize melanins in conidia (Aramayo and Timberlake, 1990). Activity of Lcc increases
after harvest of the L. edodes fruiting body, and a Lcc in L. edodes purified from fruiting
bodies after harvest can oxidize DOPA (Nagai et al., 2003). Therefore, it is considered that
Tyr and Lcc are involved in melanin synthesis after harvest of L. edodes fruiting bodies.

2.1 Tyr involvement in gill browning after harvest

Tyr (EC 1.14.18.1) is an ubiquitous enzyme in nature and the key enzyme in the process of
melanin biosynthesis (van Gelder et al., 1997). Tyr catalyzes oxidation of phenolic
substrates to quinone, which spontaneously polymerizes into dark-colored pigments
known as melanin in the presence of nucleophilic moieties. Tyrs are found in a wide range
of organisms, including prokaryotic and eukaryotic microorganisms, plants, invertebrates,
and mammals. Tyrs are involved in a variety of biological functions, for example skin
pigmentation in mammals and browning in plants and mushrooms. In mushrooms,
browning after harvest has been investigated, especially in A. bisporus and L. edodes.
Browning after harvest is considered a consequence of the Tyr-catalyzed oxidation of
phenolic substrates (e.g., DOPA) into quinones leading to the formation of dark pigments
of melanins. The enzymatic pigmentation of mushrooms is mediated largely by Tyr
(Jolivet et al., 1998).
Several reports revealed that Tyr is related to gill browning after harvest of L. edodes fruiting
bodies. It was reported that Tyr activity increases in the gills during postharvest
preservation (Kanda et al., 1996a). A Tyr (LeTyr) was purified and characterized as the L.
edodes Tyr, and LeTyr can catalyze tyrosine to DOPA (Kanda et al. 1996a, 1996b). The gene
encoding LeTyr (Letyr) was isolated, and anti-serum was synthesized to LeTyr (Sato et al.,
2009). Sato et al., (2009) showed that LeTyr increased after harvest, suggesting that LeTyr
catalyzes DOPA synthesis for melanin formation after harvest in L. edodes. Letyr is the only
Tyr encoding gene in L. edodes (Sato et al., 2009). On the other hand, Tyr is involved in
browning of the surface of vegetative mycelia (Sano et al., 2010), suggesting that LeTyr is
involved in melanin synthesis in different tissues. Browning of the surface of vegetative
mycelia is regulated by light, and the blue light receptor PHRB regulates Letyr (Sano et al.,
2009). This suggests that melanin synthesis in vegetative mycelium under light is caused by
an increase of LeTyr via PHRB. PHRB is a homolog of WC2 in Neurospora crassa, and
expression of the WC2 homolog in L. edodes is low in the L. edodes fruiting body after harvest
(Sakamoto et al., 2009). This suggests that expression of Letyr in the fruiting body after
harvest is regulated by transcription factor(s) other than PHRB.
86 Food Quality

2.2 Lcc involvement in gill browning after harvest

White-rot fungi produce several isoforms of extracellular lignin degrading enzymes,
including lignin peroxidase, manganese peroxidase, and Lcc. These lignin degrading
enzymes are considered as secreted enzymes, but several Lccs are intracellular enzymes.
Two Lccs (Lcc1 and Lcc2) have been purified from L. edodes (Nagai et al., 2002 and 2003),
an Lcc-encoding gene has been cloned and characterized, and six Lcc encoding genes
(lcc1-lcc6; lcc1: AB035409; lcc2: AB035410; lcc3: AB046713; Lcc4: AB446445; Lcc5:
AB543788; Lcc6: AB543787) have been deposited in the DNA data bank (DDBJ) (Zhao and
Kwan, 1999; Sakamoto et al., 2008, 2009; Yano et al., 2010). Lcc1 is secreted from
vegetative mycelia in culture (Nagai et al., 2002) and is encoded by lcc1 (Sakamoto et al.,
2008). Lcc2 is expressed in the brown gills of fruiting bodies after harvesting (Nagai et al.,
2003). The putative amino acid sequence of lcc4 includes identical amino acid sequences to
the N-terminal amino acid sequence of an enzymatically digested peptide of Lcc2
(Sakamoto et al., 2009). We expressed the lcc4 gene heterologously in Aspergillus oryzae,
and observed Lcc activity of the recombinant enzyme (Yano et al. 2009). These data
suggest that lcc4 encodes Lcc2, as designated by Nagai et al. (2003), and the gene lcc2
(Accession No. AB035410) does not. Therefore, the Lcc2 purified by Nagai et al. (2003) is
designated Lcc4 in this paper. Expression of lcc1 is high in vegetative mycelia (Sakamoto
et al., 2008), but lcc4 is expressed only in the fruiting body and increases after harvest
(Sakamoto et al., 2009). Furthermore, Lcc1 cannot oxidize DOPA (Nagai et al., 2002), but
Lcc4 can (Nagai et al., 2003; Yano et al., 2009). These observations collectively suggest that
Lcc4 is involved in melanin synthesis after harvesting by catalyzing L-DOPA to DOPA
quinone. Gill browning after harvesting of L. edodes fruiting bodies is considered to be
caused by melanin synthesis due to cooperation of LeTyr and Lcc4 and their increased
levels after harvest.

3. Cell wall degrading enzymes involved in fruiting body autolysis

Fruiting body softening occurs due to cell wall degradation. The cell wall of L. edodes is
constructed of several polysaccharides, such as β-1,3-glucan, β-1,6-glucan, chitin, and
chitosan (Shida et al. 1981). Thus, β-1,3-glucanase, β-1,6-glucanase, chitinase, chitosanase
are involved in cell wall degradation after harvesting. Cell wall degrading enzymes found
in the L. edodes fruiting body after harvest will be introduced. One of the cell wall
components, β-1,3-1,6-glucan, called lentinan, is used for antitumor therapy. Lentinan
degradation after harvesting is caused by an increase in β-1,3-glucanase activity after
harvesting. Thus, controlling β-1,3-glucanase expression is very important to keep the
lentinan content in the L. edodes fruiting body after harvest. There are few reports on cell
wall degrading enzymes, such as endoglucanase and exoglucanase encoding in
basidiomycetous fungi, such as in A. bisporus. However, there were fewer reports on
glucanases related to senescence of mushrooms when we started our research on
senescence in L. edodes. There were several studies on chitinase activity in several
mushrooms, but there was no evidence of a relationship between chitinases and
mushroom senescence at that time. Cell wall degrading enzymes in L. edodes fruiting
bodies after are discussed in this section.
Senescence of the Lentinula edodes Fruiting Body After Harvesting 87

3.1-glucanases
The cell wall structure of fungi is constantly changing during mycelial growth and the cell
cycle. Morphological changes involving synthesis, reorienting and lysis of the cell wall
structure are an essential process in fungi (Enderlin and Selitrennikoff, 1994; Seiler and
Plamann, 2003). The cell wall forms a multilayered complex of polysaccharides,
glycoproteins and proteins with cross-linkages. The polysaccharides consist of β-glucans
(mainly β-1,3-glucan and β-1,6-glucan), chitin, chitosan, mannans and -glucans
(Aimanianda et al., 2009; Fontaine et al., 2000; Kollár et al., 1997). Fungi such as ascomycetes
and basidiomycetes produce enzymes associated with these polysaccharides. Some of these
fungal glycoside hydrolases (GH) act on cell wall components and are responsible for
morphological changes (Adams, 2004; Fukuda et al., 2008; Mahadevan and Mahadkar, 1970;
Wessels and Niederpruem, 1967).
Most basidiomycetes form a fruiting body (mushroom) for sporulation. The cell walls of the
fruiting body are constructed mainly from chitin, β-1,3-glucan and β-1,6-glucan. Several cell
wall polysaccharides extracted from basidiomycetes, such as Schizophyllum commune (Tabata
et al., 1990), Agaricus blazei (Ohno et al., 2001), Coprinopsis cinerea (Bottom and Siehr, 1979),
Grifola frondosa (Ishibashi et al., 2001) and L. edodes (Chihara et al., 1969), show bioactive
(antitumor) activity, and some of these antitumor compounds are β-1,3-glucans with β-1,6-
linked branches, i. e., lentinan isolated from L. edodes (Chihara et al., 1969) and schizophyllan
from S. commune (Ooi and Liu, 2000). Studies of these mushroom polysaccharides have
indicated that their content decreases during storage after harvest, suggesting that
polysaccharides of the cell wall are self-degraded by enzymes associated with cell wall
autolysis during fruiting body senescence (Minato et al., 2004). Two types of exo-β-1,3-
glucanases, EXG1 (Sakamoto et al., 2005a) and EXG2 (Sakamoto et al., 2005b), and two endo-
β-1,3-glucanases, TLG1 (Sakamoto et al., 2006) and GLU1 (Sakamoto et al., 2011) have been
reported in L. edodes fruiting bodies. EXG1 is classified in GH family 5 and EXG2 is classified
in GH family 55. TLG1 is not classified in the GH family, so far, and TLG1 is similar to
thaumatin-like protein (Sakamoto et al. 2006). GLU1 is classified in a new GH family, GH128
(Sakamoto et al. 2011).
β-1,6-glucan is thought to be a unique and essential component of fungal cell walls. Whereas
β-1,3-glucan forms a microfibrillar structure, β-1,6-glucan forms a branched amorphous
structure (Kollár et al., 1997). Lichen Umbilicaria species produce a linear glucan composed
of only β-1,6-linkages (pustulan; Nishikawa et al., 1970). Many fungi are considered to
secrete β-1,6-glucanases, some of which have been purified and characterized (Bryant et al.,
2007; Oyama et al., 2002; Moy et al., 2002; Pitson et al., 1996). All of the isolated enzymes are
β-1,6-glucan endohydrolases (EC 3.2.1.75) classified into GH family 5 or 30 of the GH-A clan
in the CAZy database. Konno and Sakamoto (2011) first reported a β-1,6-glucanase in
basidiomycetes, which is classified in the GH30 family, from L. edodes fruiting body.

3.1.1 exo-β-1,3-glucanase
Several exo--1,3-glucanases genes of fungi have been isolated and characterized. In
Saccharomyces cerevisiae, three exo-β-1,3-glucanase encoding genes (EXG1, EXG2, SSG1) have
been cloned and characterized (Larriba et al., 1995). The EXG1 gene encodes two main
extracellular exo-β-1,3-glucanases (Kuranda and Robbins, 1987; Vázquez de Aldana et al.,
1991), EXG2 encodes an exo-β-1,3-glucanase attached to the plasma membrane (Correa et al.,
88 Food Quality

1992; Larriba et al., 1995) and SSG1 (also known as SPR1) encodes a sporulation-specific exo-β-
1,3-glucanase (Muthukumar et al., 1993). Several other exo-β-1,3-glucanase genes have been
isolated from yeasts, including Candida albicans (Chambers et al., 1993), Kluyveromyces lacis,
Hansenula polymorpha and Schwanniomyces occidentalis (Esteban et al., 1999). In filamentous
fungi, an exoglucanase encoding gene, EXG2, was isolated from the plant pathogenic fungus
Cochliobolus carbonum (Kim et al., 2001). In basidiomycetous mushrooms, there is little
information about exo--1,3-glucanases encoding genes such as a report of two open reading
frames (ORF) for exo-β-1,3-glucanase sequences in A. bisporus (van den Rhee et al., 1996).
Sakamoto et al., (2005b) purified and characterized an exo-β-1,3-glucanase from L. edodes,
designated EXG1. EXG1 is a β-1,3-glucanase classified in the GH5 family. EXG1 has high
similarity to exo-β-1,3-glucanase sequences in A. bisporus (van den Rhee et al., 1996). EXG1 in
L. edodes is specifically expressed in fruiting bodies but not in vegetative mycelia, and
decreases after harvesting. EXG1 can degrade a β-1,3-glucan, laminarin, but not lentinan
(Sakamoto et al., 2005a; Table 1). These observations suggest that EXG1 is not involved in
lentinan degradation or fruiting body senescence. On the other hand, EXG1 is expressed
abundantly in growing stipes. Cell wall degrading and rearranging enzymes are important for
stipe elongation; therefore, EXG1 could have a function in stipe elongation in L. edodes fruiting
bodies.

1) Sakamoto et al. 2005a; 2) Sakamoto et al. 2005b; 3) Sakamoto et al. 2006; 4) Sakamoto et al. 2011; 5)
Konno and Sakamoto 2011
a) not degraded b) degraded
Table 1. Summary of glucanases (EXG1, EXG2, TLG1, GLU1, and PUS30) purified from L.
edodes.

Several ascomycetous fungi, including Aspergillus saitoi (Oda et al., 2002), and the
mycoparasitic fungi Trichoderma harzianum (Cohen-Kupiec et al., 1999) and Ampelomyces
quisqualis (Rotem et al., 1999) express exo--13-glucanases other than the GH5 type of exo-β-
1,3-glucanases. These exo--13-glucanases are classified in the GH55 family. An second exo-
β-1,3-glucanase EXG2 was purified and characterized from L. edodes fruiting body after
harvest (Sakamoto et al. 2005b). EXG2 is expressed in growing stipe but only weakly
Senescence of the Lentinula edodes Fruiting Body After Harvesting 89

expressed in fresh gills. Expression of EXG2 increases immediately after harvest, and then
becomes abundant in fruiting bodies 3 days after harvest. EXG2 has a high ability for
degrading lentinan (Sakamoto et al. 2005b; Table 1), producing mainly glucose and
gentiobiose (Fig. 2A). This suggests that EXG2 can hydrolyze β-1,3-glucan linkages in spite
of the existence of a β-1,6-glucan linkage in lentinan (Fig. 2A). This hydrolyzing activity is
demonstrated in a GH55 enzyme in Phanerochaete chrysoporium (Ishida et al., 2010). These
observations suggest that EXG2 in L. edodes is mainly related to lentinan degradation after
harvest. EXG2, like EXG1, is also abundantly expressed in growing stipes. This suggests that
EXG2 has a dual function in stipe elongation and fruiting body senescence.

3.1.2 endo-β-1,3-glucanase
A. bisporus produce an endo-β-1,3-glucanase (Galán et al., 1999) and an endo--1,3-glucanase
has also been reported from L. edodes (Grienier et al., 2000) that exhibits similarities to the
anti-fungal thaumatin-like (TL) proteins that are highly conserved in plants. Plants
accumulate a large number of pathogenesis-related (PR) proteins, which are divided into
five families (PR1-PR5), and TL proteins share sequence homology with the thaumatin
isoforms from arils of Thaumatococcus danielli (Dudler et al., 1994) are members of the PR5
family (van Loon and van Strien, 1999). Some TL proteins exhibit both -1,3-glucan binding
(Tundel et al., 1998), and endo--1,3-glucanase activities (Grenier et al., 1999). Proteins with
endo--1,3-glucanase activities in mushrooms that have glucan binding activity were
purified (Grienier et al., 2000), and their N-terminal amino acid sequences were similar to TL
proteins. Sakamoto et al. (2006) purified an endo--1,3-glucanase, TLG1, and isolated the
encoding gene tlg1, which has high similarity to TL proteins. TL protein encoding genes
have been found in organisms outside of the plant kingdom, such as in the nematode
Caenorhabditis elegans (Kitajima and Sato, 1999) and in the locust Schistocerca gregaria
(Brandazza et al., 2004). It is reported that genes with similarity to TL proteins are highly
conserved in fungi (Sakamoto et al., 2006). Fungal TL proteins also exhibit both -1,3-glucan
binding and -1,3-endoglucanase activities (Grenier et al., 2000). Expression of TLG1 in L.
edodes is significantly weak in vegetative mycelia, growing stipes and fresh fruiting bodies
(Sakamoto et al. 2006). TLG1 is only expressed abundantly in fruiting bodies after harvest.
TLG1 has lentinan and cell wall degrading activity (Sakamoto et al., 2006; Table 1),
suggesting that TLG1 is specifically involved in lentinan degradation and fruiting body
senescence.
GLU1 was purified as an endo--1,3-glucanase from L. edodes fruiting bodies after harvest,
and was separated from TLG1. GLU1 showed greater hydrolyzing activity against laminarin
than against lentinan by endo lytic manner (Fig. 2A; Sakamoto et al., 2011). The gene
encoding GLU1 was isolated (glu1), and recombinant GLU1 in Pichia pastoris had endo--
1,3-glucanase activity (Sakamoto et al., 2011). However, GLU1 did not have significant
similarity to known -1,3-glucanases or to any glycoside hydrolases. Endo--1,3-glucanases
are divided into two classes, EC 3.2.1.6 [endo-1,3(4)--glucanase] and EC 3.2.1.39 (endo-1,3-
-glucanase), based mainly on substrate specificity. Because GLU1 did not degrade -1,3-
linkages within -1,3-1,4-glucans such as barley glucan, the enzyme was categorized into EC
3.2.1.39. A phylogenetic tree was drawn based on sequences; GLU1 obviously does not
belong to existing GH families containing EC 3.2.1.39 glucanases on the CAZy server
(Sakamoto et al., 2011). Moreover, amino acid sequence analysis of GLU1 revealed no
significant homology with any previously described functional proteins, the enzyme and
90 Food Quality

other similar proteins were classified in a new GH family, GH128. Expression of glu1 is
weak in vegetative mycelia and growing fruiting bodies, but increases after harvest (Fig. 3).
Its expression pattern is very similar to TLG1

Fig. 2. A Degradation of β-1,3-1,6-glucan (lentinan) by EXG2 and GLU1. Left standards (St.)
indicates β-1,3-glucan oligosaccharides, and right St. indicates β-1,6-glucan oligosaccharides.
B Combination of GLU1 and EXG2 from L. edodes on the β-1,3-glucan hydrolysis. Pachyman
(1%, w/v) (lane 2, untreated substarate) was firstly treated with GLU1 (lane3), and the
GLU1-treated pachyman was further incubated with of EXG2 (lane 4). Standards (lane 1) are
glucose (Glc), laminaribiose (Lam2), laminaritriose (Lam3) and Laminaritetraose (Lam4).

Fig. 3. Expression pattern of the glu1 a: transcription levels of the glu1. 1: mycelium from
liquid culture. 2: young fruiting bodies under 1 cm. 3: young fruiting bodies, 1-2 cm. 4: stipe
of young fruiting bodies, 2-3 cm. 5: pileus of young fruiting bodies, 2-3 cm. 6: stipe of young
fruiting bodies, 3-5 cm. 7: pileus of young fruiting bodies, 3-5 cm. 8: gill of mature fruiting
body; 9: gill of fruiting body at 1 day after harvest; 10: gill of fruiting body at 2 days after
harvest; 11: gill of fruiting body at 3 days after harvest; 12: gill of fruiting body at 4 days
after harvest. b: Western blot analysis of GLU1 after harvest. D0, D1, D2, D3 and D4 indicate
day 0 (fresh fruiting body), day 1 day 2, day 3, day 4 after harvesting, respectively. c:
Western blot analysis of GLU1 after spore dispersal. D0, D5, D10, D15 indicates day 0, day 5
day 10, day 15 after cap veil open.

(Sakamoto et al. 2006). GLU1 can degrade lentinan, suggesting that GLU1 is involved in
lentinan degradation after harvest. Enzymatic properties and expression pattern of GLU1 and
Senescence of the Lentinula edodes Fruiting Body After Harvesting 91

TLG1 are very similar; therefore, it is considered that these two enzymes have a redundant
function in senescence of the L. edodes fruiting body.
It has been reported that some of the enzymes in the GH16 family have endo--1,3-
glucanase activity. One GH16 family gene, mlg1, was found in fruiting bodies after harvest
(Sakamoto et al., 2009). Furthermore, several other GH16 family genes, mlg2 (DJ432070),
ghf16.1 (DJ432068), and ghf16.2 (DJ432069) were isolated from L. edodes fruiting bodies.
However, the enzymatic activities of the proteins encoded by these genes are not known;
therefore, there is no clear evidence for a relationship between GH16 family enzymes and
autolysis of the L. edodes fruiting body so far.

3.1.3 endo-β-1,6-glucanase
Some mycoparasitic fungi such as Trichoderma species produce an extracellular β-1,6-
glucanase, member of GH30, for attack and degradation of host cell walls during their
mycoparasitic action (De la Cruz and Llobell, 1999; Djonović et al., 2006; Montero et al.,
2005). However, little information is known about the physiological function and role of the
fungal β-1,6-glucanases. Sakamoto et al. (2009) found one GH30 protein, ghf30 is up-
regulated after harvest. Furthermore, LePus30A, an endo-type -1,6-glucan hydrogenase,
and classified as a member of GH family 30, was purified from L. edodes (Konno and
Sakamoto 2011). LePus30A was the first purified basidiomycetous protein characterized as a
GH 30 member (Konno and Sakamoto 2011). LePus30A has high levels of similarity to
proteins from basidiomycetous species such as L. bicolor, S. commune and C. cinerea,
suggesting that -1,6-glucanases widely conserved in basidiomycetous fungi. The transcript
level of lepus30a in fruiting bodies undergoing postharvest preservation for 2-4 days was
also significantly higher than at other stages of the life cycle. This result supported an
important role for LePus30A in the degradation of the cell wall's complex structure during
fruiting body senescence after harvest. LePus30 has no activity toward lentinan, but the
enzyme showed activity for the cell wall glucans from L. edodes fruiting bodies (Konno and
Sakamoto 2011). In addition, LePus30A degrades cell wall glucans producing glucose and -
1,6-linked oligoglucosides. Therefore, LePus30A is mainly contribute to a degradation of the
-1,6-glucan rich content, during fruiting body senescence. The expression of lepus30a was
also observed in mycelium and young fruiting body. This result implicates that the -1,6-
glucanase also contributes in hyphal growth and branching, or development (Djonović et al.
2006; Moy et al. 2002).

3.1.4 lentinan degradation

Cell wall degrading enzymes, namely exo--1,3-glucanase EXG2 and endo-β-1,3-glucanases
TLG1 and GLU1 are involved in lentinan degradation after harvesting (Table 1). To study
the cooperation of L. edodes endo- and exoglucanases during -1,3-glucan hydrolysis,
pachyman (a linear -1,3-glucan from the basidiomycete Poria cocos) hydrolysates obtained
after treatment with GLU1 were further treated with EXG2, and the products were analyzed
(Fig. 2B). As a result, formed various length of oligosaccharides through the endo-type
manner of GLU1 were further degraded to glucose and disaccharide (laminaribiose) by the
exo-type manner of EXG2 (Fig. 2B). Thus, the cell wall after harvesting of L. edodes fruiting
92 Food Quality

bodies appear to be degraded by combination of endo-type glucanases (GLU1 and TLG1),

and exo--1,3-glucanase (EXG2). GLU1 and TLG1 were suggested to have similar biological
functions in the process of lentinan degradation after harvest. LePus30A showed its high
substrate specificity for a linear-1,6-glucan polysaccharide (pustulan) and very low activity
toward a branched -1,3/1,6-glucan (laminarin). Since mainly glucose was liberated when
laminarin was treated with LePus30A, the enzyme presumably cut the -1,6-linked side chains
in laminarin (DP range 20-30, -1,3/1,6=7/1). TLG1 and GLU1 shorten main chain length of
lentinan, therefore, side chain of partially degraded lentinan would be cut by PUS30A. After
all, lentinan is degraded immediately after harvest by cooperation of increased EXG2, TLG1,
GLU1, PUS30A after harvest to oligo-saccharides and glucose (Fig. 4).

3.2 Chitinase
As chitin is one of the fungal cell wall components, chitinase and chitin synthase are very
important for morphogenesis of fungi. There are several reports on chitinase activities in
basidiomycetous mushrooms (for example, Kamada et al. 1981), but few on the relationship
between chitinase and fruiting body senescence. Higher chitinase activity is observed in
fruiting bodies following harvest compared to the activity just after harvest (Fig. 5). Genes
encoding chitin degrading enzymes were identified in fruiting bodies after harvest (chi1,

Fig. 4. Scheme of degradation of -1,3-1,6-glucan after harvest of the L. edodes fruiting body
Senescence of the Lentinula edodes Fruiting Body After Harvesting 93

chi2; Sakamoto et al., 2009). The putative amino acid sequences of chi1 and chi2 contain a
motif found in the GH18 family (Fig. 6). These genes do not have significant similarity to
known chitinase encoding genes, but have significant similarities to hypothetical genes in
basidiomycetous genomes such as S. commune, Serpula lacrymans and Postia placenta.
A putative chitinase encoding gene has also been found in an EST sequence from L. edodes
(Suizu et al. 2008); the cloned full-length gene, chi3, has a GH18 domain and chitin binding
domains (Fig. 6). Expression of chi3 also increased after harvest (Fig. 7). These observations
suggest that increased expression of chitinases has an important role in fruiting body

Fig. 5. chtinase activity after harvest measured by using 4-MUF-chitin (Hood 1991). The X
axis indicates 0: fresh fruiting body; 1: fruiting body at day 1 after harvest ; 2 fruiting body at
day 2 after harvest; 3 fruiting body at day 3 after harvest; 4: fruiting body at day 4 after
harvest. The Y axis indicates unit/g protein.

Fig. 6. Chitin related enzymes upregulated after harvest of the L. edodes fruiting body.
94 Food Quality

senescence after harvest. Other genes encoding putative enzymes related to chitin
modification were cloned from fruiting bodies after harvest (Sakamoto et al., 2009),
including chitin deacetylase (chd1) and chitosanase (cho1). The putative amino acid sequence
of chd1 has a polysaccharide deacetylase domain in the middle and a serine-rich region at
the C-terminus that are present in the chitin deacetylase from C. neoformans (Levitz et al., 2001;
Fig 6). The DNA sequence of the chitosanase from L. edodes, cho1, has high similarity with that
of A. oryzae, but there are no significantly similar sequences in the basidiomycetous genome
databases available so far. Chitosan is also a cell wall component of L. edodes (Pochanavanich et
al., 2002). It was reported that chitin deacetylase (chd1) and chitosanase (cho1) were
upregulated after harvesting of the L. edodes fruiting body (Sakamoto et al., 2009). The
observation that the chi1, chi2 and cho1 genes do not have significant similarity to other
basidiomycetous genes suggests that L. edodes has a unique chitin and chitosan metabolism
system. These data indicate that the cell wall of the L. edodes fruiting body is possibly degraded
as a result of increased glucanase and chitinase activity following harvest.

4. Regulation of senescence related genes

As shown above, many genes involved in fruiting body senescence increased after harvest
of the L. edodes fruiting body, including those for phenol oxidases, involved in gill browning,
and cell wall degrading enzymes involved in autolysis after harvest. Expression of many
other genes increased after harvest of the L. edodes fruiting body (Sakamoto et al., 2009). The
functions of these genes in senescence of the fruiting body are still unclear, but certainly
gene expression is drastically changed after harvest. Transcription factors or chromatin
remodelling could cause these drastic changes in gene expression. A gene encoding a
putative transcription factor, exp1, was isolated from L. edodes fruiting bodies after harvest.
The putative amino acid sequence of exp1 displays high similarity with the sequence for exp1
from C. cinerea (Accession No. AB363984; Muraguchi et al., 2008), and contains two HMG
boxes in the C-terminus. Proteins that have HMG boxes have functions in transcription
factor or chromatin remodeling. In this section, genes other than phenol oxidases and cell
wall degradation enzymes, but expressed after harvest will be discussed.

4.1 Unknown genes increased after harvesting

Expression of numerous genes increases after harvest of L. edodes fruiting bodies, but their
functions in senescence are still unknown. For example, genes encoding riboflavin forming
enzyme (baw28) and malate dehydrogenase (mdh) were isolated from L. edodes fruiting
bodies. The putative amino acid sequence of baw28 gene displays 53% identity to the C.
albicans riboflavin forming enzyme and 33% identity to the L. edodes riboflavin forming
enzyme (Accession No. AB116639) that is expressed specifically in the fruiting body (Hirano
et al., 2004). The putative amino acid sequence of baw28 has a barwin-like endoglucanase
domain in the N-terminus, and a serine-rich region in the C-terminus. The mdh gene
identified in this study does not contain any significant motifs, but displays 34% identity to
Aspergillus fumigatus malate dehydrogenase. Expression of the genes increased after harvest
(Fig. 7); therefore, the genes presumably have some biological function in senescence, but
their function is unclear.
Many genes upregulated after harvest in the L. edodes fruiting body have been reported but
do not have significant similarities to other genes (Sakamoto et al., 2009; Fig. 8). On the other
Senescence of the Lentinula edodes Fruiting Body After Harvesting 95

hand, genome sequence information in basidiomycetous fungi is increasing, so extensive

new information has been found from unknown genes. One of them, a gene that has
similarity to glucoamylase (Fig. 8 amy). Expression of amy1 is weak in vegetative mycelia
and young fruiting bodies, but significantly increases after harvest. The ghf79 has similarity
to GH79 family which includes enzymes glucronidase. The gene lup33 has high similarity to
hypothetical proteins found in basidiomycetous genomes. The function of the hypothetical
proteins is unclear, but expression of the gene specifically increases in the L. edodes fruiting
body after harvest. On the other hand, there are several genes that have no similarity to any
other genes, including hypothetical proteins in the basidiomycetous genomes available so
far. These genes are L. edodes specific genes. Expression of these L. edodes unknown protein
encoding genes, lup23, lup410, lup66, and lup48, increases significantly after harvest
(Sakamoto et al., 2009, Fig. 8), but is weak in vegetative mycelia and young fruiting bodies.
The function of these genes is still unknown, but these genes would have a specific role in
senescence of the L. edodes fruiting body after harvest.

Fig. 7. Comparison of expression levels of baw28, mdh and chi3 in fresh fruiting bodies and
fruiting bodies (D0) at day 3 (D3) after harvest. The Y axis represents the ratio of mRNA
levels of each gene to that of gpd.

4.2 putative senescence related gene transcription regulating factor exp1

Putative transcription factors, exp1 which is up-regulated after harvest are isolated from L.
edodest. As the mRNA level of exp1 was higher three days after harvest than on the day of
harvest, exp1 is likely to be involved in fruiting body senescence after harvesting. The L.
edodes exp1 gene, a homolog of exp1 isolated from C. cinerea (Muraguchi et al., 2008), contains
two HMG boxes in the C-terminus. The HMG1/2 class proteins have been considered
architectural components of chromatin that have a general role in the regulation of
chromosomal functions (Thomas et al., 2001). Several proteins that have an HMG box are
considered to be transcription factors, or have been shown to interact with transcription
factors (Wissmüller, 2006).
C. cinerea opens the cap of its fruiting body by lysing lamellae in the cap during spore
diffusion, and the C. cinerea exp1 mutant cannot open its cap (Muraguchi et al., 2008). This
suggests that exp1 in C. cinerea controls cap autolysis during spore diffusion by regulating
the genes that encode cell wall lysing enzymes. For example, expression of tlg1, which is a
homolog of one of the cell wall degrading enzymes for senescence in L. edodes, is
suppressed
96 Food Quality

Fig. 8. Expression pattern of unknown genes found in the L. edodes fruiting body. The Y axis
represents the ratio of mRNA levels of each gene to that of gpd. The X axis indicates 1:
mycelium from liquid culture. 2: young fruiting bodies under 1 cm. 3: young fruiting bodies,
1-2 cm. 4: stipe of young fruiting bodies, 2-3 cm. 5: pileus of young fruiting bodies, 2-3 cm. 6:
stipe of young fruiting bodies, 3-5 cm. 7: pileus of young fruiting bodies, 3-5 cm. 8: gill of
mature fruiting body; 9: gill of fruiting body at 1 day after harvest; 10: gill of fruiting body at
2 days after harvest; 11: gill of fruiting body at 3 days after harvest; 12: gill of fruiting body
at 4 days after harvest
Senescence of the Lentinula edodes Fruiting Body After Harvesting 97

in the C. cinerea exp1 mutant (Fig. 9). Cell wall lysis after spore diffusion is observed in L.
edodes, and expression of the cell wall degrading enzymes EXG2, TLG1 and GLU1 increases
after spore diffusion (Sakamoto et al., 2005b, 2006, Fig. 3). Expression of these cell wall
degrading enzymes also increased after harvest, suggesting that the systems for cell wall
lysis in the fruiting body after harvesting and after spore diffusion are similar in L. edodes.
These results suggest that exp1 in L. edodes might control senescence of the fruiting body by
regulating genes that are expressed after harvesting, such as genes that encode cell wall
degrading enzymes.

Fig. 9. expression pattern of the tlg1 in exp1. The Y axis represents the ratio of mRNA
levels of the tlg1 to that of gpd.

5. Genes downregulated after harvesting

It has been reported that a large number of genes downregulated after harvest are likely
involved in normal fruiting body formation and are needed to maintain freshness of the
fruiting body (Sakamoto et al. 2009). Translation, transcription and protein metabolism
related genes, in addition to those involved in spore formation, cytoskeleton, and cell cycle
are downregulated after harvest, and many more transcription factors are also
downregulated after harvest (Sakamoto et al. 2009). Interestingly, genes identified among
those upregulated after harvest are completely different from those in the downregulated
genes. This suggests that gene transcription is drastically altered after harvesting of the
fruiting body.

5.1 Transcription, translation, and protein metabolism related genes

Many transcription, translation and protein metabolism related genes are down-regulated
after harvesting. Several RNA related genes downregulated after harvest, such as the
Pumilio family RNA binding protein, RNA binding protein 5-like protein, ATP-dependent
helicase and pre-mRNA splicing factor (Sakamoto et al., 2009). These RNA related proteins
are involved in transcription and translation (de Moore et al., 2005; Rogers et al., 2002);
therefore, expression of many genes might be changed after harvesting as a result of
suppression of these transcription and translation related genes. Heat shock proteins (i.e.,
Hsp70 and Hsp90), chaperonin and calnexin, which are involved in the proper translation
and folding of proteins to control protein quality (Saibil, 2008; Spiess et al., 2004; Williams,
2006), were also down-regulated. This suggests that the protein quality control system in the
98 Food Quality

L. edodes fruiting body is less effective following harvest. Hsp70 and Hsp90 were especially
highly expressed in young fruiting bodies (Fig. 10); therefore, these proteins have an important
function in normal fruiting body development. Several proteases and peptidases, such as
aspartic protease (pro1, pep1), metallopeptidase MepB and gamma-glutamyltranspeptidase
were downregulated after harvest (Sakamoto et al., 2009), and proteasome-related protein
encoding genes such as proteasome 26S, proteasome 26S subunit alpha type 6 and proteasome
26S ATPase subunit 3 were also downregulated after harvest (Sakamoto et al., 2009). These

Fig. 10. Putative transcription factors found in the L. edodes fruiting body. The Y axis
represents the ratio of mRNA levels of each gene to that of gpd. The X axis indicates 1:
mycelium from liquid culture. 2: young fruiting bodies under 1 cm. 3: young fruiting bodies,
1-2 cm. 4: stipe of young fruiting bodies, 2-3 cm. 5: pileus of young fruiting bodies, 2-3 cm. 6:
stipe of young fruiting bodies, 3-5 cm. 7: pileus of young fruiting bodies, 3-5 cm.
Senescence of the Lentinula edodes Fruiting Body After Harvesting 99

proteins comprise a proteasome complex that is involved in protein degradation (Wolf and
Hilt, 2004). Decreased protease and proteasome activity would extend the life of proteins
expressed after harvest. These data suggest that transcription, translation and posttranslational
protein metabolism are drastically altered in fruiting bodies after harvest.

5.3 Mitosis and meiosis related genes

Many mitosis and meiosis related genes are down-regulated after harvesting. Several
meiosis related proteins, such as a meiotic recombination related protein encoding gene,
dmc1, show altered expression in harvested fruiting bodies (Sakamoto et al., 2009). DMC1 is
a recombinase that is required for DNA pairing during recombination. Two DMC1-
encoding genes were previously identified in C. cinerea, namely Rad51 (Stassen et al., 1997)
and LIM15 (Namekawa et al., 2005). Whereas Rad51 is involved in both mitosis and meiosis,
LIM15 is specifically involved in meiosis, being expressed during meiosis and disappearing
immediately after meiosis (Nara et al., 1999). The putative amino acid sequence encoded by
dmc1 has significant similarity to LIM15/DMC1 in C. cinerea. The dmc1 gene was found to be
transcribed abundantly in the gills of fresh fruiting bodies and was downregulated after
harvesting (Sakamoto et al., 2009). These data suggest that dmc1 is specifically involved in
meiosis, and is downregulated after spore formation. Expression of the cell division related
genes septin and cdc48 reportedly decreased after harvest (Sakamoto et al., 2009). Both of
these genes have important functions during cell division (Lindsey and Momany, 2006;
Cheeseman and Desai, 2004). Septins are GTPases that form filaments in fungi and
animals and are involved in membrane trafficking, coordinating nuclear divisions, and
organizing the cytoskeleton (Lindsey and Momany, 2006). cdc48 has a variety of cellular
functions; for instance, it is implicated in the membrane fusions that occur after mitosis to
reassemble the endoplasmic reticulum and the Golgi apparatus (Cheeseman and Desai,
2004). Genes encoding the cytoskeleton related proteins actin and beta-tubulin were also
downregulated after harvest (Sakamoto et al., 2009). These genes are essential for
progression of cytokinesis (Nanninga, 2001). This suggests that the frequency of cell
division decreases after harvest.

5.4 Putative transcription factors identified by reverse subtraction

Several putative transcription factors are downregulated after harvest of the L. edodes
fruiting body. One putative transcription factor gene, hlh1, which contains a basic helix-
loop-helix motif involved in DNA binding (Fig. 11), was also cloned (Sakamoto et al., 2009).
Basic helix-loop-helix proteins are a group of eukaryotic transcription factors that exert a
determinative influence on a variety of developmental pathways (Littlewood and Evan,
1995). The putative amino acid sequence of hlh1 contains a nuclear localization signal and
displays high similarity to a hypothetical protein from C. cinerea. The hlh is specifically
expressed in young fruiting bodies (Fig. 10). Expression of ftf1, ftf2, ftf3, which have a fungal
specific transcription factor domain (Fig. 11), was observed in L. edodes fruiting bodies
(Sakamoto et al., 2009). The putative amino acid sequences of these genes have a similar
structure to the priB gene product, which is involved in fruiting body development in L.
edodes (Miyazaki et al., 2004). The priB gene is transcribed specifically in the L. edodes fruiting
body, and regulates fruiting body-specific genes (Kaneko et al., 2001; Miyazaki et al., 2004).
100 Food Quality

The ftf1 ftf2, and ftf3 are highly transcribed in fresh fruiting bodies and their mRNA levels
decreased after harvesting (Fig. 10; Sakamoto et al. 2009), suggesting that the genes regulates
genes involved in fruiting body development, but does not regulate genes involved in
fruiting body senescence.

Fig. 11. Putative transcription factors found in the L. edodes fruiting body

The putative protein product of hsf1 has no significant similarity to other known proteins,
but has significant similarities to hypothetical proteins in several basidiomycetous fungi,
such as C. cinerea and Serpula lacryman. The putative amino acid sequence of hsf1 has a helix-
turn-helix DNA binding domain for binding to a heat shock element (Fig. 11) that is
conserved in the upstream region of heat shock proteins (Pirkkala et al., 2001). Its expression
pattern was different from heat shock proteins (Fig. 10); therefore, hsf might not directly
regulate the expression of heat shock proteins. The putative transcription factor, zin1 and
zin2, were also highly transcribed in fresh fruiting bodies, with a decline in expression after
harvesting (Fig. 10; Sakamoto et al. 2009). The zin1 and zin2 gene products contain a zinc
finger motif (Fig. 11). The putative amino acid sequence of zin2 has a MYND-type zinc
finger motif as well as significant similarity with the zf-MYND domain containing protein in
L. bicolor that is upregulated by infection with ectomycorrhiza (Martin et al., 2008), but there
is no significant similar sequence to the zin2 gene sequences in other basidiomycetous
genomes available so far. In contrast, there are other basidiomycetous proteins that have a
MYND domain; for example, Fuz1 from U. maydis contains an MTND domain and is
Senescence of the Lentinula edodes Fruiting Body After Harvesting 101

involved in cell morphogenesis (Chew et al., 2008). These results suggest that zin2 is
involved in fruiting body development. zip is also specifically expressed in fruiting bodies
but not in vegetative mycelia (Fig. 10). The putative amino acid sequence of zip1 has a
leucine zipper DNA sequence, which is found in several transcription factors, and also has
an Aft osmotic stress domain (Fig. 11). The zip does not have any significant similarity to
known transcription factors but has significant similarities to hypothetical proteins in
several basidiomycetous fungi, such as C. cinerea and S. lacrymans.
A homolog of the blue light receptor white collar 2 (WC2) in N. crassa was also found in L.
edodes fruiting bodies. In N. crassa, WC2 acts as a component of the blue light receptor by
interacting with white collar protein 1 (WC1). WC2 in L. edodes was cloned and
characterized; the protein has been designated PHRB; Sano et al., 2009). Genes similar to
WC1 have also been reported in L. edodes (PHRA; Sano et al., 2008) and in C. cinerea (dst1;
Terashima et al., 2005). The C. cinerea dst1 mutant cannot form a mature cap under normal
light/dark conditions, and the shape of the fruiting body of the dst1 mutant is very similar
to that formed by the wild-type mushroom when grown in complete darkness (Terashima et
al., 2005). Since WC2 and PHRA in L. edodes interact with each other, they could act as a blue
light receptor in L. edodes that regulates fruiting body development in the light (Sano et al.,
2010). Gene disruptionof the WC2 homolog in C. cinerea results in a similar phenotype to
that of dst1 (Nakazawa et al. 2011).
These genes might not be directly involved in senescence of the L. edodes fruiting body after
harvest, but these putative transcription factor genes presumably would be involved in
normal fruiting body morphology. A decrease in expression of the putative transcription
factor genes would influence morphology after harvest.

6. Future perspectives
As shown above, numerous genes are related to senescence of the L. edodes fruiting body
after harvest. However, the mechanism of senescence of the L. edodes fruiting body after
harvest is not fully understood. To understand the mechanism involved, an understanding
of total gene expression changes after harvest is needed. Transcriptome analysis such as
microarray and SAGE analysis is useful to understand total gene expression changes. In
Lentinula edodes, there are several reports of transcriptome analysis, such as an EST study
(Suizu et al., 2009) and SAGE analysis (Chem 2011). For SAGE analysis, genomic sequence
data are needed to increase accuracy. Public genomic sequence data of L. edodes are not yet
available so far, but genomic sequencing will be easier than before using a next-generation
genome sequencer, such as the Illumina GA IIx. Furthermore, SAGE analysis is optimized
for next-generation sequencing to obtain a larger amount of sequence tagged data
(Matsumura et al. 2003). Therefore, gene expression profile changes after harvesting of the L.
edodes fruiting body will be revealed in more detail by using a combination of genome
sequence analysis and Super-SAGE (Matsumura et al. 2003) with a next-generation
sequencer. In C. cinerea, autolysis after spore dispersion is similar to that of L. edodes, and
public genomic sequence data of C. cinerea are available, so it is possible to compare gene
expression profiles during autolysis in C. cinerea and L. edodes. Such research will provide a
basic understanding of mushroom senescence.
102 Food Quality

To prove the function of genes, gene disruption or gene silencing studies will be needed.
Gene disruption systems have been constructed in several mushrooms such as C. cinerea
(Nakazawa et al. 2011). A homologous gene recombination system has also been constructed
(Irie et al. 2003), but a gene disruption system by homologous recombination has not yet
been constructed in L. edodes. However, gene silencing by RNAi has succeeded in L. edodes
(Nakade et al., 2011). Therefore, genes upregulated after harvest will be knocked down by
RNAi, which will reveal the functions of the genes, such as exp1 and exg2, in senescence.
This research will provide useful data for breeding strains with fruiting bodies that remain
fresh for a longer period after harvest.

7. Conclusion
After harvesting of the L. edodes fruiting body, drastic gene expression changes occur. In
particular, expression of phenol oxidases (Tyrs and Lccs) and cell wall enzymes (glucanases
and chitinases) increase after harvest. This suggests that a gene regulation system for
senescence exists in L. edodes. exp1, which is a putative transcription factor or chromatin
remodeling related protein, is one of the candidates for regulation of the drastic gene
expression changes involved in senescence. The process would include programmed cell
death, but there are few studies on the relationship between programmed cell death and
mushroom morphology. Therefore, studies on senescence in mushrooms will be important
not only for applied agricultural science, but also for basic science.

8. Acknowledgment
The authors thank Dr. Kamada, Okayama University and Dr. Muraguchi, Akita prefectural
University for giving us the exp1 mutant and related information in C. cinerea. The authors
also thank to previous members of Microorganism lab. in Iwate biotechnology Research
Center.

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4913
 7

Feeding Habits of Both Deep-Water

Red Shrimps, Aristaeomorpha foliacea and
Aristeus antennatus (Decapoda, Aristeidae)
in the Ionian Sea (E. Mediterranean)
Kostas Kapiris
Hellenic Centre for Marine Research,
Institute of Marine Biological Resources,
Greece

1. Introduction
1.1 The deep-water fishes’ diet habits: Some notes on the present and the future
knowledge
The study of dietary composition and feeding habits of fish has been considered as one of
the most important topics in the ecology of animal marine communities. Both the biology of
species (as a population level) and their interaction with biological and environmental
factors are influenced by the food resources exploited by fish.
At bathyal depths, most of our knowledge on deep-sea demersal communities, mainly for
both fishes and large invertebrates – e.g. decapods crustaceans, comes principally from areas
where deep-water fisheries are commercially developed. Most deep-sea (>1000 m) fish are
active predators, regarded as generalist (i.e. highly diversified diets) and depend mainly on
benthopelagic and mesopelagic prey (Mauchline & Gordon, 1986). The distribution of these
preys is closely associated with the bottom; however, many demersal fishes feed principally
on vertically migrating mesopelagic organisms such as myctophids and cephalopods.
Crustacean zooplanktivores constitute the majority of deep-sea pelagic fish species and
families examined. Less common are predators that primarily ingest soft-bodied or
gelatinous zooplankton, gastropod molluscs and polychaete worms. These categories of
predators are generally represented by a few individual species within different families
(Randall & Farrell, 1997)
Many deep-sea fish show diel and seasonal feeding patterns (e.g. Macpherson, 1980;
Mauchline & Gordon, 1984; Atkinson, 1995), which have been often related to the temporal
vertical migrations of their mesopelagic prey (Gartner et al., 1997). To understand temporal
(e.g. daily, seasonal) and spatial changes in the diet and trophic habits of bathyal fish it is
crucial simultaneous sampling of their trophic resources (Madurell, 2003). However despite
the accepted role of this fauna in the bathyal food webs, little is known about their
interactions and their dynamics.
112 Food Quality

To date, studies on the feeding habits of deep-sea fish have focused mainly on depth related
changes and only few studies have addressed aspects of seasonal or diel feeding cycles.
Studies on food resource partitioning are scarce in non-littoral marine environments, with
some studies performed in deep-sea communities at a community level and within a single
feeding guild (e.g. Carrassón & Cartes, 2002). In general, food is considered as the major
limiting factor in the functioning of deep-water ecosystems and trophic aspects have been
considered as the most important factor on deep-sea faunal community organization
(Jumars & Gallagher, 1982).
The knowledge concerning the deep-sea organisms’ biology remains almost in its infancy.
Many aspects about their diet are still speculative. Many lags exist concerning the type of
the bottom, the spatial and temporal distribution of many fishes and the taxonomic
composition of the fauna. The result of further detailed studies could offer important
elements to our learning about the taxonomic composition, life history and physiology
studies, such as feeding habits (Randall & Farrell, 1997).

1.2 Feeding habits in Decapod crustaceans of shallow and deep-sea waters – General
aspects
The much diversified diet of penaeids (shrimps or prawns) was first described by Williams
(1955) who studied the stomach content of prawns from the eastern United States. Generally
speaking, the condition of the gut contents prevents the use of the most methods of
quantifying diet. Techniques such as weighting the food, measuring the size of prey or
reconstituting prey cannot be used (Dall et al., 1990). Decapods can catch mobile free-
swimming prey; the remains of fish and squid form a major part of the diet of several
species and their preferable animal food is other small crustaceans, polychaets, mollusks
(Dall et al., 1990), plant remains (Kuttyamma, 1974) and a very small portion of bacteria
(Moriarty & Barclay, 1981).
Most shrimps spend the day buried in the substratum and emerge and feed at night. Because
of their small stomach, they feed several times each night in order to obtain sufficient food.
Under natural conditions, where food items are small and dispersed, searching and ingestion
probably continue through the night and rates of ingestion and ejection are probably similar
(Dall, 1968). This allows the foregut to be filled repeatedly and enables the shrimps to take in
considerably more food if they fed only once per night (Dall et al, 1990).
Decapods can even be dominant in some deep-water regions, including the deep
Mediterranean, where subtropical species dominate in terms of biomass (Cartes & Sardà,
1992). It has been suggested that this dominance is due to the low metabolic rates of this
taxon, which has low feeding rates and hence is better adapted to live under oligotrophic
conditions than, for instance, fishes (Cartes & Sardà, 1992). In bathyal ecosystems, decapods
occupy a variety of ecological niches and exhibit a similar range of trophic levels to fish
(Polunin et al., 2001). They also have a wide variety of feeding habits or guilds (Cartes et al.,
2002), ranging from deposit feeders to carnivores, the latter including specialized species
preying on benthos (e.g. Crangonidae: Lagardère, 1977) or macrozooplankton (e.g.
Pandalidae: Cartes, 1993a). As a consequence, decapods are an ideal group in which to
analyze changes in the structure and dynamics of ecosystems exposed to temporal (e.g.
Seasonal) or spatial (e.g. depth) gradients (Cartes et al., 2007).
Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 113

2. Mediterranean deep sea waters

The Mediterranean Sea – Mare Nostrum, Mare Internum or Mediterraneum is a relatively small,
deep basin that is surrounded by continents. It is 4000 km in length and is located between
30o and 46o N and 5.50o W and 36o E (excluding the Black Sea). The Mediterranean Basin is
subdivided by a series of tranverse ridges with a north-south trend. These ridges exist in the
western (Alboran, Balearic, Tyrrhenian basins) and the eastern part (Ionian, Aegean,
Levantine) (Sardà et al., 2004 and references herein).
Deep-sea ecosystems include the waters and sediments beneath approximately 200 m depth
(Emig & Geistdoerfer, 2004) and represent the world’s largest biome, covering more than
65% of the earth’s surface and including more than 95% of the global biosphere. The deep-
sea domain of the Mediterranean is divided in three zones: bathyal, abyssal and hadal
(Table 1). The edge of the continental shelf is the boundary between the neritic (inshore)
domain and the deep-sea oceanic (offshore) domain. Its depth varies with the ocean or Sea.
The deepest point in the Mediterranean, 5,121 m, is found at the North Matapan-Vavilov
Trench, Ionian Sea. The deep-sea floor includes regions characterized by complex
sedimentological and structural features: (a) continental slopes, (b) submarine canyons, (c)
base-of-slope deposits, and (d) bathyal or basin plains with abundant deposits of
hemipelagic and turbidity mud’s (Danovaro et al., 2010).

Table 1. Comparison of the ranges in depth of the deep-sea zones of the World Ocean and
the Mediterranean Sea (Emig & Geistdoerfer, 2004).

A further unique feature of the Mediterranean is that it is one of the few warm deep-sea
basins in the world, where temperatures remain largely uniform at around 12,5-14,5ºC at all
depths, with high salinity (38,4-39,0 PSU) and high oxygen levels (4,5-5,0 ml, Hopkins,
1985). The constant temperature and salinity regime of the Mediterranean contrasts with the
Atlantic at comparable latitudes, where temperature decreases and salinity increases with
depth.
114 Food Quality

The Eastern and Western Mediterranean display important geological and biological
differences. First, the Eastern Mediterranean is geologically more active, because it is a
contact zone between 3 major tectonic plates: African, Eurasian and Arabian. The Western
Mediterranean is relatively featureless in comparison, although still not devoid of unique
environments. Biologically, the Western Mediterranean, whilst still oligotrophic by North
Atlantic standards, has relatively high primary production, especially in the Gulf of Lions,
due to the river Rhone runoff and wind mixing. The Eastern Mediterranean has very low
primary production. The existing deep Mediterranean appears to be much younger than
any other of the world’s deep ocean and only a small fraction of specialized taxa exists in its
deep-sea fauna. Traditionally the Mediterranean Sea is one of the most intensively
investigated areas of the world in both terrestrial and coastal marine biodiversity, but it lags
other regions of the world in studies of its deep-sea fauna.

2.1 Mediterranean deep sea fauna

The Mediterranean appears to be a remarkable natural laboratory for the study of the
processes of recent colonization as related to the unique history of each of the two great
Mediterranean basins (western and eastern) (Emig & Geistdoerfer, 2004) and, due to the
presence of few endemic species, is a important centre of evolution. Its fauna is composed
mainly of primitive taxonomic groups among the phyla represented, whereas a small
fraction of specialized taxa exists in the deep-sea fauna. Despite its small dimensions (0,82%
of the ocean surface), the basin hosts more than 7,5% of global biodiversity (Bianchi & Morri,
2000). However, this information is almost completely confined to coastal ecosystems, and
data on deep-sea assemblages are still limited (e.g. Ramirez-Llodra et al., 2009).
The stable environment of the deep Mediterranean Sea permits to the biotic (e.g. trophic)
factors may have a comparatively strong influence on the ecology and biology (e.g. food intake
and reproduction) of deep-Mediterranean species (Madurell & Cartes, 2006). Also, the depth
overlap between and the depth range inhabited by megafaunal fish and decapod crustaceans
(Cartes and Carrasson, 2004) are mainly explained by trophic variables (e.g. trophic level).
The deep Mediterranean fauna displays a number of characteristics that differentiate it from
other deep-sea faunas of the world’s oceans (Bouchet & Taviani, 1992): i) the high degree of
eurybathic species; ii) absence (or low representation) of typical deep-water groups, such as
macroscopic foraminifera (Xenophyophora), glass sponges (Hexactinellida), Sea-cucumbers
of the order Elasipodida, primitive stalked Sea-lilies (Crinoidea) and tunicates (Sea-squirts)
of the class Sorberacea (Monniot & Monniot, 1990); and iii) the number of endemic species
(26.6% of the Mediterranean fauna: Ruffo, 1998) declines with increasing depth, with
comparatively low endemisms below 500 m (see also Fredj & Laubier, 1985).
The existed quantitative data from this basin are scarce. Several investigations have
described low-abundance and low-diversity conditions of marine invertebrates in the
Eastern Mediterranean (Tselepides & Eleftheriou, 1992). Scientific knowledge of deep
megafaunal communities (mainly fish, crustaceans and cephalopods) was limited to the
bathymetric range exploited by fishing (down to 800-1000-m) until the early 1980’s, when
scientific expeditions began quantitatively sampling the bathyal grounds in the
Mediterranean. Such studies in the Western and Central Mediterranean have focused on the
Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 115

two most abundant groups below 600 m depth: fishes (D’Onghia et al., 2004) and decapod
crustaceans (e.g. Company et al., 2004). At depths below the 1500 m, there is an increase in
the relative abundance of crustaceans in comparison to fish (Company et al., 2004). This
change in the relative abundance of these two groups has been explained by the low food
availability at greater depths and the higher adaptation of crustaceans to low energy levels
(e.g. Company et al., 2004).

2.2 Mediterranean deep sea crustacean fauna

Decapod crustaceans are one of the dominant megafaunal groups in the deep-sea
communities of the Mediterranean Sea (Sardà et al., 1994a). The relatively oligotrophic
nature of Mediterranean waters has been presented as one of the environmental factors
contributing to the high abundance of decapod crustaceans in comparison with other
oceans, in which other megafaunal invertebrates, chiefly echinoderms, predominate (Tyler
& Zibrowius, 1992). However, though the Mediterranean is a relatively small Sea compared
with other oceans, existing data and our understanding of the continental margins at depths
below 2000 m lags behind. Decapods are much more abundant than other invertebrate
groups in the Mediterranean, in contrast to more productive oceans like the Atlantic, where
echinoderms are the dominant invertebrate group (e.g. Sardà et al., 1994a), because these
crustaceans be more competitive than other invertebrate or vertebrate megafauna in
oligotrophic environments (Maynou & Cartes, 2000).
In the W. Mediterranean 28 decapod species were identified, including 6 Dendrobranchiata,
1 Stenopodidean, 7 Caridea, 2 Thalassinidea, 2 Palinura, 3 Anomura, and 7 Brachyura.. The
most pronounced qualitative changes in the fauna were recorded between 1000 and 1200 m
and at around 2000 m. The bathyal decapod fauna mainly composed by species belonging to
the families Crangonidae, Galatheidae and Geryonidae, and the genera Nematocarcinus and
Stereomastis. In addition to this the tropical species A. antennatus, Acanthephyra eximia, and
Plesionika acanthonotus are widely distributed and frequent in the deep western
Mediterranean (Cartes, 1993b). Thirty nine decapod species have been reported in the
Eastern Ionian Sea (E. Mediterranean), of which eight were Dendrobranchiata and 31
Pleocyemata (17 Caridea, 9 Brachyura, 3 Anomura, 1 Astacidea and 1 Palinura) (Politou et
al., 2005). Concerning their depth distribution, 30 species were found in the depth zone 300-
500 m, with Parapenaeus longirostris being the most abundant species. Of the 27 species
caught in the zone 500-700 m, A. foliacea and Plesionika martia were the most abundant. In the
zone 700-900 m, 19 species were found and A. foliacea with A. antennatus were the most
numerous. Finally, the 18 decapod species encountered in the zone 900-1200 m showed low
abundance, and Sergia robusta with Polycheles typhlops predominated in numbers. From the
identified decapods, Acanthephyra eximia, Philoceras echinulatus and Pontophilus norvegicus
were mentioned for the first time in the E. Ionian Sea. Some other species, such as
Acanthephyra pelagica, Geryon longipes, Munida tenuimana, Paromola cuvieri, Parthenope
macrochelos, Pasiphaea multidentata, Plesionika narval, Polycheles typhlops, Sergestes arachnipodus
and Sergestes arcticus have been reported for the area only in the gray literature.

3. Aristeidae: Distribution and particular hydrological conditions

In updated systematic approaches (Pérez-Farfante & Kensley, 1997) both deep-sea red shrimps
- A. antennatus and A. foliacea - are the only Mediterranean representatives of the Aristeidae
116 Food Quality

family (superfamily Penaeoidea, Order: Decapoda, Sub-order: Dendrobranchiata). The species

of the family (Aristaeomorpha, Aristeus and Plesiopenaeus), all large-size commercial shrimps,
occur in deep water off the continental shelf. Important morphological characteristics of both
species are i) light exoskeletons and long pleopods suggesting good swimming ability; ii)
secondary sexual dimorphism concerning body size and the rostrum; and iii) an open
thelicum. The spermatophores are larger in A. antennatus, in relation to a greater fecundity: in
fact the females of A. antennatus produce about four times more eggs than A. foliacea females of
the same size (Orsi Relini & Semeria, 1983). The life history of the two species therefore begins
with a very different energy budget and probably body development.

3.1 Aristaeomorpha foliacea: Distribution and importance

The giant red shrimp or deep-sea red shrimp A. foliacea (Risso, 1827) is a species of a very
wide geographical distribution in the world. It occurs in the Mediterranean Sea and the
eastern Atlantic, the western Atlantic, the Indian Ocean and the western Pacific from Japan
to Australia, New Zealand and the Fiji Islands (Pérez Farfante & Kensley, 1997) Gracia et al.
(2010) recently explored deep waters off the Yucatan Peninsula in Mexico and showed that
A. foliacea represents a potential fishing resource. Nowadays, A. foliacea constitute a valuable
deep shrimp fishery off the south-eastern and southern sectors of the Brazilian coast
(Dallagnolo et al., 2009). The giant red shrimp has been recently found in large quantities in
the Colombian Caribbean Sea (Paramo & Urlich, in press) (Figure 3).
In the Mediterranean, the species is of great economic interest and, together with A.
antennatus (Risso, 1816), represents the main target species of the slope trawl fisheries down
to 800-1000 m (Demestre, 1994; Ragonese et al., 1994a,b; Sardà & Cartes, 1994b; Matarrese et
al.,1997). This species is heavily exploited in Western Mediterranean, and is currently fished
in the Central Mediterranean; its stocks are pristine in the Eastern Mediterranean (Bianchini
& Ragonese, 1994; Papaconstantinou & Kapiris, 2003; Gönülal et al., 2010) and its
exploitation is not yet been developed.

Fig. 2. Geographical distribution of A. foliacea (Source: http://www.aquamaps.org/receive.php).

Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 117

The economic importance of the giant red shrimp in the Mediterranean enhanced the
scientific interest of the study and evaluation of its stocks. Most scientific information on A.
foliacea comes from the central Mediterranean, where the species is relatively abundant and
exploited by the commercial fishery (e.g. D’Onghia et al., 1998). It concerns its biology (e.g.
D’Onghia et al., 1994; Levi & Vacchi, 1988; Mura et al., 1997), ecology (e.g. Ragonese et al.,
1994a) and fisheries (e.g. Ragonese, 1995; Matarrese et al., 1995). In the eastern
Mediterranean (Greek waters), knowledge of the species was recently obtained. Some
information on its distribution (Kallianiotis et al., 2000; Kapiris et al., 2001c), morphometry
(Kapiris et al., 2002; Kapiris, 2005) and biology (Kapiris et al., 1999 ; Kapiris & Thessalou-
Legaki, 2001b, 2006, 2009; Papaconstantinou & Kapiris, 2003), feeding (Kapiris et al.,2010)
and fishery (Mytilineou et al.,2006) appeared in the literature. According to the present state
of knowledge the species depth distribution ranges between 123 and 1047 m, with a
maximum abundance from 400 to 800 m in most areas. The maximum depth of occurrence
was found to be 1100 m for the whole Mediterranean basin (Politou et al., 2004).

Fig. 3. Specimens of A. foliacea (Source: http://www.google.gr).

3.2 Aristeus antennatus: Distribution and importance

During the last twenty years a variety of aspects of the blue-red deep water shrimp (A.
antennatus Risso, 1816) (Figure 4) have been studied in detail in the western, central and
eastern Mediterranean Sea, such as fisheries (e.g. Demestre & Martín, 1993; Bianchini &
Ragonese, 1994; Sardà et al., 1998; 2003; Papaconstantinou & Kapiris, 2001; D’Onghia et al.,
2005), biology (e.g. Kapiris & Thessalou-Legaki, 2001a, 2006, 2009; Kapiris et al., 2002;
Kapiris & Kavvadas, 2009; Matarrese et al., 1997; Sardà & Cartes, 1993; Mura et al., 1998;
Follesa et al., 1998; Orsi Relini & Relini, 1998; Sardà et al.,1998), ecology (e.g. Sardà & Cartes,
1997; Cartes & Maynou, 1998; Kapiris et al., 1999; Kapiris & Thessalou-Legaki, 2011), and
physiology (e.g. Company & Sardà, 1998; Puig et al., 2001).
In the Mediterranean, this important commercially and biologically species may be fished
from depths of 80 m along the Algerian coast at night (Nouar, 2001), with more abundant
distribution between 400 m and 800 m in Tyrrhenian waters (Aquastudio, 1996) (Figure 5).
Its eurybathic distribution ranges from 100 and 150 m to nearly 1000 m in the western Ionian
Sea (south Italy, Relini et al., 2000), down to 800 in the eastern Ionian (Papaconstantinou &
Kapiris, 2001) and 900-1000 m off Catalonia (Demestre & Martín, 1993; Sardà et al., 1998).
118 Food Quality

Nevertheless, experimental catches (Sardà et al., 2004) have been made down to a depth of
3300 m. This broad depth distribution range for this species has led to a number of
hypotheses concerning its ecology and possible relationships between the exploited
populations on the upper and middle slope and the non-exploited populations dwelling
deeper on the lower slope (Sardà et al., 2003). Its biology (reproduction, sex-ratio, feeding
habits, and population dynamics and fisheries) is relatively well known down to 800 m,
where fishery occurs.

Fig. 4. Female and male individual of A. antennatus (Source:

http://cobmedits2011.wordpress.com/produccion-scientifica/evaluaciones)

Fig. 5. Geographical distribution of A. antennatus

(Source: http://fishbase.sinica.edu.tw/slp/SpeciesSummary.php?group=All&ID=ITS-
96062).
Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 119

An increasing abundance gradient from the western to the eastern Mediterranean is

confirmed by several previous works for these both deep-sea red shrimps. The abundance of
A. foliacea increases gradually eastwards, from the Tyrrhenian Sea to the Straits of Sicily and
the waters around Greece, where it becomes more abundant than A. antennatus (Politou et
al., 2003). Different hydrological conditions (i.e. temperature and salinity) between the
westernmost and the easternmost areas have been reported to affect the species distribution
(Relini & Orsi Relini, 1987). A. foliacea is considered to be linked to warmer and more saline
water masses than the other deep Sea shrimp A. antennatus (Ghidalia & Bourgeois, 1961).
Furthermore, the eastern Mediterranean deep water transient (Klein et al., 1999) may play a
role in the increased abundance of A. foliacea in the eastern Ionian Sea, since this event is
associated with a significant upward nutrient transport, which is most pronounced in the
eastern Ionian Sea, and may result in greater biological productivity.

4. Diet studies on Aristeids mainly in the E. Mediterranean – The aim of this

study
Both aristeids present an increased diversity in their diet (Burukovsky, 1972; Lagardère,
1977; Relini Orsi & Wurtz, 1977; Cartes, 1994, 1995; Gristina et al., 1992; Maurin & Carries,
1968; Chartosia et al., 2005). ). Brian (1931), for the first time, has studied the alimentary
habits of A. foliacea and A. antennatus in the Ligurian Sea and stressed the big diversity of
prey types consumed by the two shrimps (e.g. pelagic, benthic and benthopelagic
organisms). In the Ionian Eastern Sea the diet and the feeding habits of both aristeids have
been studied in details (Kapiris & Thessalou-Legaki, 2011; Kapiris et al., 2010).
The object of this chapter is to provide a detailed description of the feeding habits of both
deep-water red shrimps in the Eastern Ionian, in relation to Season, size and sex. New
information concerning its feeding patterns provides greater insight concerning the
population ecology of these important and unexploited resources of the Greek Seas. In
addition, such data could serve in the comparison of its life history traits along the
Mediterranean.

4.1 The study area

Few studies have been carried out about the dominant demersal fish species found on the
upper-middle slope (between 473 and 603 m) in the Ionian Sea (e.g. D’Onghia et al., 1998;
Kallianiotis et al., 2000; Labropoulou & Papaconstantinou, 2000; Madurell et al., 2004). The
results of those studies indicated that the Ionian demersal ichthyofauna is similar to the
other eastern Mediterranean areas. Dominant species in the eastern Mediterranean, such as
H. mediterraneus, and C. agassizi which are plankton feeders, are rare (or absent) in the
Catalan Sea (Stefanescu et al., 1994). Macrofauna from the eastern Mediterranean decrease in
biomass below 400m and there is a significantly lower biomass of meiofauna than in the
Western Basin (Tselepides & Eleftheriou, 1992; Danovaro et al., 1999). These low levels of
benthos biomass may reinforce the dominance of top predators feeding on planktonic
resources in the Ionian Sea.
Exploratory sampling of A. foliacea and Aristeus antennatus took place along the south coast
of the Greek Ionian Sea, between Zakinthos Island and Peloponnisos Peninsula (Figure. 6).
A total of 92 hauls were taken during 12 experimental trawl survey cruises on a monthly
120 Food Quality

basis (December 1996–November 1997). Samples were collected by the commercial trawler
Panagia Faneromeni II (26 m in length, 450 HP) using a net with a cod-end mesh size of 18
mm from knot to knot. The results of the feeding habits and diet of both aristeids in the
Eastern Ionian are given below.

80
0m
Epirus

A A

80
0m
Ion
B

ian
Se
a

Pelop
Salami na Isl

Zakinthos Egina Isl.

on isso
80
0m
# #

s
#
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lop
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##
## on
iss
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os
ia n

###
Zakinthos
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200m ##
#

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500m
Mediterrannean Sea 800m

Fig. 6. Study area in the Eastern Greek Ionian Sea

4.2 foliacea’s feeding habits

4.2.1 Feeding activity and food quality
The highly diversified diet observed in A. foliacea is typical of bathyal penaeoideans in the
Western Mediterranean (Cartes, 1995). The feeding activity of A. foliacea in the Eastern
Ionian Sea was examined studing the stomach fullness according the equations (i) wet food
weight (g) per 100 g shrimp wet weight [% body weight (BW) Wet = (SWW⁄BW) x 100] and
(ii) dry food weight (g) per 100 g wet weight [%BWDry = (SWD⁄BW) * 100] (Héroux &
Magnan, 1996). The nutritional quality (food quality) of the preys has been estimated by two
ways: (a) % dry weight (DW) = (SWD⁄SWW) 100 and (b) % ash free dry weight (AFDW) =
(AFDW⁄SWD) x 100, where SWW=stomach wet weight, g, SWD= stomach dry weight, gr
after 24 h of oven drying at 70o C), ash-free dry weight (AFDW; as loss on ignition at 450o C
for 3 h) and BW is the body weight. All the weights were measured to an accuracy of 0.0001
g). The food quality indices are a measure of total organic matter and form a better
estimation of food value that wet weight, which includes substantial amounts of inorganic
material (Hiller-Adams & Childress, 1983). The stomach fullness of A. foliacea varied
Seasonally in both sexes and both fullness indices (%BW Wet, %BW Dry) were significantly
higher in females than in males for each Season. The maximum values of %BW Wet in both
sexes occurred in winter and the minimum in spring.
Diet quality (%BW Dry and %AFDW) also differed significantly among Seasons for both
sexes of A. foliacea. In general, few significant differences in food quality were detected
between males and females for each Season. Males and females of A. foliacea presented the
highest values of both quality indices in spring and the minimum in winter.
Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 121

The feeding habits of this decapods identified in the Ionian Sea are generally comparable to
those reported in other regions of the Mediterranean, such as in the Catalan Sea (Cartes,
1995), Sicilian Channel (Gristina et al., 1992) and Aegean Sea (Chartosia et al., 2005). Any
difference observed in the whole Mediterranean, such as food diversity, different food
categories and mean number of prey could be due to bottom morphology (Cartes 1995) and
to the oligotrophic conditions of the Eastern Mediterranean. This characteristic of the
Eastern Mediterranean could also explain the increased number of pelagic prey consumed
by A. foliacea compared to the western part of the basin (Cartes, 1995). The considerably
higher water temperature of the Eastern Mediterranean (Politou et al., 2004) may also play a
role, resulting in a higher metabolic rate of this species, in comparison with those from the
western part of the basin.
Trophic diversity ((H’, Shannon-Wiener index) varied slightly among Seasons in both sexes
(Figure 7) and no statistically significant differences were established between sexes. The
maximum diversity (3.00 and 3.04 for males and females, respectively) and mean number of
prey items (2.9 and 3.1 for males and females, respectively) were found in summer for both
sexes of A. foliacea.
The observed low number of empty stomachs [(number of empty stomachs per number of
stomachs examined) * 100] (Hyslop, 1980) in the present study, ranging from 4,5 to 18,1%,
indicating either a high feeding rate or slow digestion rate, could be explained by their high
metabolic rates. The lowest proportion of empty stomachs of A. foliacea was found in spring
for both sexes, followed by summer. In contrast, the highest number of empty stomachs was
found in autumn for females and summer for males.

Shannon Diversity

AUTUMN

SUMMER
Season

SPRING

WINTER

1,600 1,800 2,000 2,200 2,400 2,600 2,800 3,000 3,200

A. foliacea, males A. foliacea, females

Fig. 7. Diversity index (H’, Shannon-Wiener index) values for A. foliacea per sex and season
in the Ionian Sea.

In general, a decrease in diversity and mean prey items with increasing overlap was
observed. In the Eastern Ionian Sea, the giant red shrimp fed on a greater proportion of
pelagic resources and prey with a good swimming ability, such as the natantian decapods,
122 Food Quality

and to a lesser extent on benthic prey, indicating that this shrimp is an active and effective
predator of the bathyal zone in the Eastern Mediterranean. The characteristic of its active
predation could be also confirmed by the very low abundance of infaunal and epibentic
prey (e.g. polychaetes, bivalves and gastropods) in the stomachs of this species. The
increased abundance of fishes and cephalopods in their foreguts most probably reflects the
great scavenging ability of this species. In any case, this does not exclude the possibility that
this species feeds actively upon fishes and cephalopods.

4.2.2 Food habits in relation to sex, season and size

The diets of both sexes of A. foliacea consisted of 60 different prey categories (most as
species-level prey categories). The preys belonged chiefly to three major groups: (i)
crustaceans – particularly decapods, reptantia (anomurans, brachyurans), amphipods,
euphausiids, ostracods, copepods, mysids, tanaidaceans, cumaceans, (ii) cephalopods and
(iii) fishes. These three prey categories constituted 72–82% of the relative abundance and
total occurrence for males and 70–88% of the relative abundance and the total occurrence in
females. The most dominant natantians found were the nektobenthic Plesionika martia,
Plesionika heterocarpus and Plesionika giglioli, followed by Pasiphaea sp., Sergestes sp. and
Solenocera sp. Some appendages from Aristeus antennatus were also found mainly in female
A. foliacea. These findings could be accidental, as they were found in the sampling stations
where both species coexisted and, thus, some body appendages could have been destroyed
and mixed during the net tow (net feeding). It is also possible that the smaller individuals of
each species were consumed by larger adults of the other, due to their voracious character,
but further study of this hypothesis is required. Among cephalopods, the dominant species
were Abraliopsis pfefferi, Pyroteuthis margarifera and Abralia veranyi. For fishes, specimens of
Myctophidae and Macrouridae were the most abundant in the foreguts.
Only a partial differentiation in the feeding behaviour, in terms of both diet composition and
feeding activity, was observed between sexes of A. foliacea. In general, both sexes fed upon
natantian decapods, particularly Plesionika spp., Sergestes sp., Pasiphaea sp., and fishes
throughout the year, while ‘other crustaceans’ and polychaetes were ingested on a secondary
basis. The consumption of the same prey items, but in different abundance and occurrence,
may be attributed to sexual dimorphism and to size difference between the sexes.
In general, the existence of regular Seasonal rhythms in the feeding activity of deep water
species is related mainly to Seasonal fluctuations in various factors including the abundance
of their prey, depth, local geographical characteristics, submarine canyons, bottom type,
Seabed features, Seasonal horizontal or diurnal vertical migrations, etc. (Cartes 1993, 1998).
In the Eastern Ionian Sea the Seasonal feeding habits of the giant red shrimp seem to be
related to reproduction, and perhaps to other biological processes, and food availability.
High observed values of trophic overlap between Seasons for both sexes indicated that
Season is not the main factor affecting the diet of deep-water shrimps in the Eastern Ionian
Sea. In spite of this, most feeding activity values (empty stomachs, quality indices, mean
number of prey items found into the stomachs, diversity index) support the finding that
feeding activity increased during spring–summer for both sexes. This increase could be
attributed to the increased reproductive activity (gonad maturity, egg-laying) observed in
this period (Papaconstantinou & Kapiris 2001, 2003). In addition, copulation begins at the
Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 123

end of winter and by spring almost all females are inseminated (Kapiris, 2004). The
minimum value of the stomach fullness in spring, in combination to the highest food quality
value and the lowest vacuity index in females in the same Season, suggests that egg
maturation is connected to the feeding habits of A. foliacea. During winter, A. foliacea had the
highest stomach fullness, but with decreased food quality. This increase of food
consumption by the giant red shrimp of the Ionian Sea during the pre-reproductive period
has also been observed in A. antennatus off the Balearic Islands. Increased feeding rates
could be the main reason for its egg development and could allow earlier gonad maturity
(Cartes et al., 2008a).
Besides the Seasonal feeding adaptation to the biological requirements (reproductive
process), food availability also plays an important role for these species in the Eastern Ionian
Sea. The highest densities in the suprabenthic fauna (mysids, cumaceans, amphipods,
isopods, tanaidaceans) have been observed during spring, but zooplankton (chiefly
copepods, ostracods and chaetognaths) were more abundant in summer and autumn. Such
fluctuations in food availability have also been shown in the diets of both sexes of A. foliacea
in this study. Thus, the diet of the giant red shrimp probably reflects localized forage
assemblages rather than a preference for specific items.
The size-related changes in diet composition are an important factor in determining
ecological relationships of marine organisms during their life span. Comparison of diet
composition, dietary diversity, and feeding activity among small, medium and (only for
females) large individuals reveals that this decapod undergoes slight changes in feeding
habits with increasing body size, as well as gonad maturity, in the Eastern Ionian Sea. Small
males and females (immature individuals) consumed fewer prey due to their smaller
stomachs, with more frequent occurrence of epibenthic prey in their foreguts. Larger,
mature individuals of both sexes are more efficient predators due to their greater swimming
ability and larger mandibles. A positive trend of ingesting larger prey with increased size
was observed only for females. This is the first time where this gradation, probably due to
the population structure and to morphological variation among size classes and sexes, has
been observed for A. foliacea. In general, somatic growth and gonad development induce a
change in this species’ feeding behavior as the body grows an increase in the mean weight
of prey and a decrease in the mean number of prey items per stomach was obvious.
However, almost the same prey occurred in the stomachs of small, medium and large
specimens, but in different proportions

4.3 Aristeus antennatus’ feeding habits

4.3.1 Feeding activity and food quality
A differentiation has been presented in A. antennatus diet according to the depth in the
western Mediterranean (Cartes, 1994), the feeding time (Cartes, 1993a) and the daily
consumption of food (Maynou & Cartes, 1997, 1998; Cartes & Maynou, 1998). The diet of A.
antennatus changed as a function of depth at around 1000 m depth in the Catalan Sea, as a
function of Seasonality influences by planktonic prey in deeper zones and by possible
nocturnal movements upward along the slope canyons (Cartes, 1993a, 1994; Cartes et al.,
2010). The importance of spatial patterns in its diet and feeding habits and the main
environmental variables controlling these trophic aspects has been studied by Cartes et
124 Food Quality

al.(2008b) in Western Mediterranean. In the whole E. Mediterranean, the feeding habits of A.

antennatus have been studied in detail in the Ionian (Kapiris & Thessalou-Legaki, 2011) and
the Aegean Sea (Chartosia et al., 2005).
The observed low number of empty stomachs in the Greek Ionian (mean value of the empty
stomachs in males was 6,53 and for females was 8,54) could be explained by their high
metabolic rates (Company, 1995). Significant statistical differences amongst the Seasonal
medians of both fullness indices were found [%BW Wet (for both sexes) and %BW Dry (only
in females)]. The maximum values of %BW Wet were determined in winter in both sexes
and the minimum in spring. Both fullness indices were statistically higher in females than
those of males (Figure 8).
Significant statistical differences amongst the Seasonal medians of both indices of food
quality (%DW, %AFDW) were established only for females. Females presented a lower
value of %DW and higher of %AFDW than males, in spring, while their highest values of
both quality indices were found in spring (Figure 8).

Females

60 0,25

50
0,2

40
Food quality

0,15

Fullness
30
0,1
20

0,05
10

0 0
Winter Spring Summer Autumn
Season

%DW % AFDW % BW Wet % BW Dry

Males

60 0,12

50 0,1

40 0,08
Food quality

Fullness

30 0,06

20 0,04

10 0,02

0 0
Winter Spring Summer Autumn
Season

%DW % AFDW % BW Wet % BW Dry

Fig. 8. Seasonal values of stomach fullness and food quality of both sexes of A. antennatus in
the Greek Ionian Sea.
Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 125

The diet of A. antennatus both sexes consisted of 54 prey categories. These prey items
belonged mainly to smaller crustaceans (e.g. natantian decapods, Plesionika sp., Sergestes sp.,
euphausiids, tanaidaceans), molluscs primarily gastropods, bivalves, polychaetes
(Eunicidae, Spionidae, and Nereididae), chaetognaths and, to a lesser extent, fishes. The
above prey categories consisted of 71–82% of the relative abundance and total occurrence for
males and 61–81% of the relative abundance and the total occurrence in females. Its
diversified diet in the present study area consists of increased endobenthic and epibenthic
invertebrates and includes organisms that are related with the Seabed, nekton and
decapods. This species is among the few megabenthic predators whose diet is mainly based
on benthos in the deep Mediterranean (Cartes & Carrassón, 2004). The increased abundance
of gastropods, echinoderms, polychaetes — chiefly Eunicidae, sipunculans and
chaetognaths in the stomachs, confirms that this species in the Greek Ionian Sea could be
considered a “slow hunter”, foraging mainly on organisms that live completely or partially
buried in the substratum. The macrophyte consumption was rare in both sexes and
probably reflects availability in the marine environment.
The data of the present study confirm that A. antennatus could be considered a less active
and slower hunter than the other aristeid species (A. foliacea) found in the same area (Kapiris
et al., 2010) and preys on detrivores or small predators occupying a lower position in the
benthopelagic food chain (Maynou & Cartes, 1997). The feeding activity patterns of A.
antennatus in the Greek Ionian Sea are, more or less, comparable to those reported in other
geographical regions, such as the central (e.g. Relini & Orsi Relini, 1987; Follesa et al., 2009)
or in the western Mediterranean (Cartes & Sardà, 1989; Maynou & Cartes, 1998). Apparent
differences in the activity patterns should be attributed to the more oligotrophic character of
the Ionian Sea (E. Mediterranean) in relation to the western one and to the bottom
morphology (Cartes, 1995). The above mentioned oligotrophic character of the eastern
Mediterranean could explain the presence of the increased number of some pelagic preys in
its stomachs, in comparison to the western one (Cartes, 1995), but – as we said before – these
preys constitute the minority comparing to the benthic ones. Some remains of the sympatric
A. foliacea in the stomachs of A. antennatus and vice versa could be accidental, since they
have been found in the sampling stations where both species coexisted and, thus, some
body appendages were destroyed and mixed during the net tow (net feeding). It is possible
that the smaller individuals of each species, due to their voracious character, can be fed by
the adults of the other one. In any case, further study is necessary.
Only a partial differentiation in the feeding behaviour between sexes, in terms of both diet
composition and feeding activity, is observed. Males exhibit lower values of fullness, food
quality indices and evenness than females. Both sexes consume the same prey items, but in
different abundance and occurrence. From the above results, a slightly higher predatory
ability of females is shown. These differences could also be attributed to sexual dimorphism
and to size difference between the sexes.

4.3.2 Seasonal differences

Taking into consideration the narrow depth sampling range, the estimated values of the
trophic overlap indicate that, Season could not be considered as the main factor affecting the
diet of blue–red shrimp in the Greek Ionian Sea, like in A. foliacea. Notwithstanding, some
particular topics are analyzed below. The existence of regular Seasonal rhythms in the
126 Food Quality

feeding activity of deep-water species is mainly related to the Seasonal fluctuations of

abundance of prey they consume, the depth, the local geographical characteristics, the
submarine canyons, the type of bottom, the Seabed, the Seasonal horizontal and diurnal
vertical migrations, etc. (Cartes, 1993a, 1998). In addition to this, the Seasonal changes in
stomach fullness of blue–red shrimp could be possibly linked to the oceanographic
processes and to the several water masses, at least in the W. Mediterranean (Cartes et al.,
2008b; Maynou, 2008).
The above slight Seasonal changes in the feeding dynamics of this aristeid in the Greek Ionian
Sea seem to be related mainly to their biological processes (e.g. mating and reproduction) and
to the food availability. The increased values of food quality indices and diversity support the
finding that feeding activity seemed to increase qualitatively – in the period spring–summer,
mainly for females. In addition to this, the observed highest empty stomachs found in these
Seasons, mainly for females, could be attributed to the increased volume of the gonads which
press the stomach. This increase of the highly energetic diet could be attributed to the
increased pre- and reproductive activity observed in this period (Kapiris and Thessalou-
Legaki, 2006, 2009). As Cartes et al. (2008a) noted A. antennatus seemed to increase the energy
intake in its diet from February to April-June in the western Mediterranean. During winter
both sexes of A. antennatus in Greek Ionian Sea consume an increased number of prey items,
having as a result the highest stomach fullness, but of decreased quality. This phenomenon
could be related to the mating period which takes place in this Season (Kapiris & Thessalou-
Legaki, 2006, 2009). Besides the Seasonal feeding adaptation to the biological requirements, the
food availability also plays an important role for this species in the Greek Ionian Sea. Madurell
& Cartes (2005) point out that, in the same study area, the suprabenthos fauna (mysids,
cumaceans, amphipods, isopods, and tanaidaceans) showed the highest densities in spring,
while the zooplankton fauna (chiefly copepods, ostracods and chaetognaths) was more
abundant in autumn and summer. In agreement with the results of the present study, the
above fluctuations of food availability are also shown in the diet of A. antennatus. Thus, the
diet of the blue–red shrimp probably reflects localized forage assemblages rather than a
preference for specific items. In addition to this, these results reinforce the opinion concerning
the “accidental hunting” of A. antennatus.

4.3.3 Ontogenetic differences

Comparison of the diet composition, dietary diversity and feeding activity between the
small size, medium size and – only in females – large size individuals reveals that this
species undergoes changes in feeding habits with increasing body size and gonad maturity
in the Greek Ionian Sea. Small immature individuals consume less prey, mainly epibenthic,
– but of increased quality – due to their smaller stomach. Larger mature specimens of both
sexes are more efficient predators because of their greater swimming ability and their larger
mandibles. The positive trend between increasing females' body size and consumption of
larger prey is observed could be attributed to the population structure and to the
morphological characteristics of the different size classes and sexes. In general, somatic
growth and gonad development induce a change of A. antennatus feeding behaviour in the
Greek Ionian Sea: as the body grows, an increasing mean weight of prey and mean number
of prey items per stomach was obvious. However, almost the same prey occurred in the
stomachs of small, medium and large specimens, but in different proportions.
Feeding Habits of Both Deep-Water Red Shrimps, Aristaeomorpha foliacea
and Aristeus antennatus (Decapoda, Aristeidae) in the Ionian Sea (E. Mediterranean) 127

5. Conclusions
Our results on the feeding ecology of both deep water shrimps could be considered as
primary importance for the future management of deep water assemblages, since they play
an important role. Since the deep waters in the E. Ionian Sea are almost unexploited, the
present data could elucidate the relationships between species in this ecosystem improving,
thus, the knowledge and the trophic relationships among the species helping in their
integrated management in the future.
According all the studies carried out on both decapods feeding habits, A. foliacea exploits
different resources from those used by A. antennatus and, despite both shrimps have similar
morphologies and size ranges, the exploitation of different resources probably both species to
coexist in the same areas (Cartes, 1995). In addition to this, since both deep-sea red shrimps
belonging in the same family, have an almost similar depth distribution It is expected that they
have similar energy values (in terms of wet mass), water body content (K. Kapiris unpublished
observations) and oxygen consumption rates (Company & Sardà 1998).
Concluding, the increased demand of the large energetic content and the food availability in
the same period make us suggest that both facts could stimulate fecundity in the deep-sea
blue–red shrimp in the E. Mediterranean. A similar trend has been shown for the same
species in the western Mediterranean (Cartes et al., 2008a, b). Generally, energy reserves
strongly affect fecundity and reproduction in fishes (e.g. Lloret et al., 2005) and have been
also observed in deep-water decapods (Fanelli & Cartes, 2008).

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