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Determination of selenium content in Irish
commercial Agaricus bisporus by Atomic
Absorption
Master Thesis
Catarina Maria Araújo Coelho
Master Degree in Consumer Sciences and Nutrition
Porto
September, 2012
Determination of selenium content in Irish
commercial Agaricus bisporus by Atomic
Absorption
Catarina Maria Araújo Coelho
Master Degree in Consumer Sciences and Nutrition
Supervisor: Dr. Luis Miguel Cunha, Associate Professor, Department of Geosciences,
Environmental and Spatial Planing , Faculty of Sciences, University of Porto, Porto,
Portugal; Requimte – Chemical Center of University of Porto, R. D. Manuel II, Apartado
55142, 4051-401 Porto, Portugal, Porto, Portugal
Co-supervisor: Dr. Jesús Maria Frias Celayeta, Lecturer, School of Food Science and
Environmental Health, Dublin Institute of Technology, Cathal Brugha, Dublin 1, Ireland
Porto
September, 2012
“The whole idea of motivation is a trap. Forget motivation.
Just do it. Exercise, lose weight, test your blood sugar, or
whatever. Do it without motivation. And then, guess what?
After you start doing the thing, that's when the motivation
comes and makes it easy for you to keep on doing it.”
John C Maxwell
ACKNOWLEGMENTS
Because this thesis not only belongs to me, but also to the many people who
have contributed to make this achievement possible, It is a pleasure to say thanks’ to
all the people who have generously shared their time and knowledge with me.
I wish to express my sincere gratitude to my supervisor, Dr. Luis Miguel Cunha
for all help and support during these two years, plus, for to make this international
experience possible. I’m grateful for the entire dedicated time to this work, whose
expertise, understanding, great efforts and patience, added considerably to my
experience. For his revision of all my work, thanks.
I would like to sincerely thank my co-supervisor Dr. Jesús Frias for receiving me
in DIT – Dublin Institute of Technology, Ireland, where this work was carried out. Thank
you for your untiring enthusiasm, support, advice and guidance throughout this project,
which this would not have been possible, thank you for the many things I learnt while
working by his side. Also my appreciation for supplying all the necessary conditions to
perform this work.
Thanks for the financial support that I received from University of Porto, in an
Erasmus Placement Agreement, without these scholarship this international experience
wouldn’t be possible.
Special thanks go to Dr. Lubna Ahemed, for all her selfless help in experimental
design, for playing strategically and showing support when it was most needed. Her
permanent help was tireless, thanks for lead me when I was felling lost.
My acknowledgment goes to Monghan Mushrooms Lda, Ireland, which supplied
mushroom samples and allowed a long visit with singular guidance to the farms and
company.
Thanks all the technical staff of DIT for their assistance and practical advice
throughout my research, who kindly attended to everything I needed in the laboratory.
To my colleagues who shared the experimental work with me Gavin Boland,
Mayte, and Alfonso.
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Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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A biggest thanks go to postgraduates student’s in DIT: Lu, Jaya, Laura Massini,
Jaffar, and a special to only Portuguese postgraduate in DIT – Sofia Reis, thanks for all
the conversations in Portuguese, for the companionship, and for your friendly lab
advices.
There are 3 persons who have made Dublin a very special place: Valter Silva,
thanks for funny trips. Jakub and Yenessis thank you both for sharing home with me it
was a funny journey. Thank you all for your friendship.
A special mention goes to Dr.José Carlos Marques, and Dr.Vanda Pereira for
inspiring me and helping me with my academic choices. Thank both of you!
Thanks to Carla (you’re a bestest ), Cristina, Carla (CSO), João, Luis and Jorge
for making my experience in Porto more colorful. Thanks for your support and patience
during the entire periods of these journey.
The following, being “so far, so close”, have contributed to my well-being: Nuno,
Filipa, Sandra, Roberto.
A final mention goes to my family, who I would like to express a heartfelt thank
you. Thanks to my parents for their endless support and faith in me over the years, for
an outstanding example of modesty and hard work. Thanks to my brother for being the
first person who I always notice of my changes and the first person to say “go ahead”.
To my untie Margarida for your tenderness and for have always cute and comfort
words, basically for always be there. To my grandmother for the kindness that always
welcomed me back home. Thanks all of my family treasures for making part of myself.
.
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Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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ABSTRACT
Selenium content was determined in 44 samples Agaricus bisporus, the most
common edible mushrooms commercially available in Ireland. The aim of this work was
to analyze and quantify selenium contents among three different types of Agaricus
bisporus: baby buttons, closed cups and flats, the last also known as Portobello
mushrooms, and also to investigate the effect of growing conditions on them. The study
comprised four factors: type of mushroom, crop type, flush order and growing house.
The quantitative determination of Selenium was carried out by analytical development
in a graphite furnace atomic absorption spectrometer (GFAAS).
The amount of selenium accumulated in the mushrooms samples studied was
in general modest. Baby buttons selenium concentrations ranged from 2.3 - 6.2 µg/g
Se Fresh Mushroom corresponding to Flush I and III respectively, and closed cups
selenium concentrations varies in a range of 2.3 – 5.4 µgSe/g Fresh Mushroom, values
also corresponding to the first and last flush respectively. Flats were found as a type of
mushroom with the lowest selenium contents. An evident effect on the selenium
concentrations among the evolution of flush number were demonstrated, i.e selenium
contents are much higher in flush III, than in flush I and II.
The importance of these mushrooms as a source of selenium is therefore
relevant.
Keywords: A.bisporus, mushrooms; selenium; graphite furnace atomic absorption
spectrometry.
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RESUMO
A quantidade de selénio foi avaliada em 44 amostras de cogumelos de uma só
espécie, Agaricus bisporus, cogumelos comestíveis e comercialmente disponíveis na
Irlanda. O objetivo deste trabalho foi analisar e quantificar o conteúdo de selénio em
três diferentes tipos de Agaricus bisporus (Baby buttons (BB), closed cups (CC) e
flats(F também designados de Portobello)) e paralelamente encontrar um efeito no
crescimento nas amostras de cogumelos. Foram quatro os fatores em estudo, tipo de
cogumelo, tipo de crop, número da frutificação (Flush) e número da casa (house) e/ou
também designado túnel onde os cogumelos foram cultivados. A determinação
quantitativa de selénio (µgSe/g cogumelo seco) foi analiticamente desenvolvida
recorrendo à espectrometria por absorção atómica com forno de grafite (GFAAS).
Em geral, a quantidade de selénio acumulada nas amostras de cogumelos
estudadas foi considerável. As concentrações de selénio para os cogumelos do tipo
baby buttons estiveram no intervalo entre 2.3 – 6.2 µg/g Se cogumelo fresco,
correspondendo estas concentrações à fortificação (Flush) I e III respetivamente. Os
cogumelos do tipo “Flats” foram o tipo de A.bisporus que apresentou uma menor
concentração de Selénio. Foi observado um efeito evidente nas concentrações de
selénio ao longo da evolução da fortificação, ou seja o conteúdo de selénio nas
fortificações (Flush) III, foi muito mais elevado do que o conteúdo de selénio da
primeira e segunda fortificação I e II.
A importância dos cogumelos enquanto fonte de selénio na dieta diária
irlandesa aparenta ser relevante.
Palavras chave: A.bisporus; cogumelos; selnénio; espectrometria por absorção
atómica com forno de grafite (GFAAS)
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ABREVIATIONS LIST
A.bisporus – Agaricus bisporus
AA – Atomic Absorption
AAS - Atomic absorption apectroscopy
AD – Anno Domini (Latin for Year of our God)
BB – Baby Buttons
CC – Closed Cups
CO2 – Carbon dioxide
DW – Dry weight
Ergo – Ergocalciferol
F – Flats
FAO – Food and Agriculture Organization of the United Nations
GC - Gas chromatography
GFAAS – Graphite furnace atomic absorption Spectrometry
GPx - Glutathione peroxidase
H1 – House number one
H16 – House number sixteen
H17 – House number seventeen
H2O2 – Hydrogen peroxyde
H2Se – Selenium hydroxide
HG – AAS – Hydride generation atomic absorption spectrometry
ICP-AES - Coupled plasma-atomic emission spectroscopy
NAA - Neutron activation analysis
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NRI - Reference Nutrient Intake
NSPs – Polysaccharides
SeCys - selenocysteine
SeP - Selenoprotein
UK – United Kingdom
US – United States
RSD – Relative Standard deviation
WHO – World Health organization
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INDEX OF FIGURE
FIGURE 1 SCHEMATIC REPRESENTATION OF AGARICUS BISPORUS FRUITING BODY
ON WHICH THE DIFFERENT TISSUES ARE INDICATED (REPRODUCED
FROM(MOHAČEK-GROŠEV, BOŽAC ET AL. 2001)). --------------------------------------------------- 21
FIGURE 2. A. BISPORUS GROWING IN A POLYETHYLENE BAG (REPRODUCED FROM
(O'GORMAN 2010)). --------------------------------------------------------------------------------------------------- 29
FIGURE 3. REPRESENTATIVE SCHEME OF MUSHROOM GROWING CYCLE (ADAPTED
FROM (MONAGHAN MUSHROOMS. (MAY 2012) ------------------------------------------------------- 30
FIGURE 4. THE INITIAL PHASE OF CREATION OF COMPOST. THIS AERATED
SUBSTRATE PREPARATION SYSTEM HAS PIPED CONCRETE FLOOR UNDER THE
SUBSTRATE THAT FORCES AIR THROUGH THE SUBSTRATE TO MAINTAIN
AEROBIC CONDITIONS DURING THE COMPOSTING PROCESS (REPRODUCED
FROM (BEYER AND EXTENSION 1997)). ------------------------------------------------------------------- 31
FIGURE 5.SELF-PROPELLED COMPOST TURNER MOVING THOUGH A COMPOST RICK
OR PILE (REPRODUCED FROM (BEYER AND EXTENSION 1997)). --------------------------- 31
FIGURE 6. REPRESENTATION OF THE LAST PHASE OF COMPOSTING. (A) HANDFUL OF
COMPOSTED SUBSTRATE SHOWING WHITE – FLECKING (“FIREFANG”)MICROBIAL
GROWTH. (B)SPAWN GRAINS USED TO SEED THE COMPOST WITH MUSHROOM
MYCELIA. SPAWN IS COOKED, STERILIZED,GRAIN COLLED, AND INOCULATED
WITH MUSHROOM MYCELIA (REPRODUCED FROM (BEYER AND EXTENSION 1997))
---------------------------------------------------------------------------------------------------------------------------------- 32
FIGURE 7. SPAWN GROWTH IN THE CASING AND ITS THICKER RHIZOMORPH GROWTH
(REPRODUCED FROM (BEYER AND EXTENSION 1997)) ------------------------------------------ 33
FIGURE 8. THE DEVELOPMENTAL STAGES OF THE AGARICUS BISPORUS FRUITING
PROCESS. (A) MYCELIUM; (B)INITIALS-CLUMPING; (C)PIN-PRIMORDIA; (D)PEA-
SIZED PIN; (E) PRE-WHITE BUTTON (REPRODUCED FROM (BEYER AND
EXTENSION 1997). ---------------------------------------------------------------------------------------------------- 34
FIGURE 9. MUSHROOM GROWING SYSTEMS (A) SINGLE-LAYER BAG GROWING. (B)
MULTI-LAYER STRUCTURE FOR GROWING SHELVES (REPRODUCED FROM
(MARSHALL 2009)). --------------------------------------------------------------------------------------------------- 36
FIGURE 10. MAIN GROWING REGIONS IN IRELAND (REPRODUCED FROM (BORD BIA
IRISH FOOD BOARD. (AUGUST 2012)) ---------------------------------------------------------------------- 38
FIGURE 11. A. BISPORUS SAMPLES FROM RIGHT TO LEFT: BABY BUTTONS (BB);
CLOSED CUPS (CC) AND FLATS (F). ------------------------------------------------------------------------- 53
FIGURE 12. SCHEMATIC EXPERIMENTAL DESIGN OF SAMPLING. FOR EACH
A.BISPORUS WERE DONE 3 DIGESTIONS AND IN EACH DIGESTION WERE DONE 3
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REPLICATES. (E.G.: D1= DIGESTION Nº 1; S1D1R1 = SAMPLE Nº1, DIGESTION Nº1
AND REPLICATION Nº1). “D” MEANS DIGESTION AND “R” REPLICATION. IN THE END
FOR EACH MUSHROOM SAMPLE WERE MADE 9 ANALYSES. --------------------------------- 54
FIGURE 13. SCHEMATIC REPRESENTATION OF MAIN SEVEN STEPS FROM THE
EXPERIMENTAL PROCEDURE. --------------------------------------------------------------------------------- 58
FIGURE 14. CALIBRATION CURVE FOR SE STANDARDS. ABSORBANCE OF THE
ANALYTE VERSUS SE CONCENTRATION AT (100, 200, 400 AND 600µG/L). ------------- 61
FIGURE 15. ESTIMATED MARGINAL MEANS FOR LOG10 |SE|, IN FRESH MUSHROOMS,
DEPICTING THE INTERACTION EFFECT BETWEEN FLUSHE ORDER AND TYPE OF
MUSHROOMS.----------------------------------------------------------------------------------------------------------- 68
FIGURE 16.BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G FRESH
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER .
---------------------------------------------------------------------------------------------------------------------------------- 69
FIGURE 17. ESTIMATED MARGINAL MEANS FOR LOG10 |SE|, IN DRY MUSHROOMS,
DEPICTING THE INTERACTION EFFECT BETWEEN FLUSHE ORDER AND TYPE OF
MUSHROOMS.----------------------------------------------------------------------------------------------------------- 71
FIGURE 18. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G DRY
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 72
FIGURE 19. INTERACTION EFFECT BETWEEN ONE FACTOR - TYPE OF MUSHROOMS
(BB, CC AND F). --------------------------------------------------------------------------------------------------------- 73
FIGURE 20. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G DRY
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 84
FIGURE 21. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G FRESH
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 84
FIGURE 22. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G DRY
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 85
FIGURE 23. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G DRY
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 86
FIGURE 24. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G DRY
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 87
FIGURE 25. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G DRY
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 87
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FIGURE 26. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G FRESH
MUSHROOM) DISTRIBUTION WITHIN IRISH A.BISPORUS MUSHROOM ACCORDING
TO FLUSH ORDER, TYPE OF MUSHROOM, TYPE OF CROP AND HOUSE NUMBER. 88
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INDEX OF TABLES
TABLE 1. RESUME OF A.BISPORUS MORPHOLOGY CHARACTERISTICS (ADAPTED
FROM(CHANG AND MILES 2004))............................................................................................. 21
TABLE 2. PROXIMATE CHEMICAL COMPOSITION (G/100 G) AND ENERGETIC VALUE
(KJ/100 G) OF AGARICUS BISPORUS, VALUES ARE EXPRESSED IN A DRY WEIGTH
(DW) BASIS. *ND – NOT DETECTED (ADAPTED FROM (BARROS, CRUZ ET AL.
2008)) ................................................................................................................................................ 26
TABLE 3. PROPERTIES AND MECHANISMS OF BIOACTIVE COMPOUNDS AND
A.BISPORUS EXTRACTS EVALUATED IN ANIMAL MODELS OR ANIMAL CELL LINES
(ADAPTED FROM (ROUPAS, KEOGH ET AL. 2012)) ............................................................. 27
TABLE 4. OPTIMUM CHARACTERISTIC OF PRIME GRADE MUSHROOM (ADAPTED
FROM(LEONARD AND STAFF 1999)). ...................................................................................... 35
TABLE 5 OVERVIEW OF GENERAL SELENIUM AMOUNTS IN ENVIRONMENT (ADAPTED
FROM (ŘEZANKA AND SIGLER 2008)) .................................................................................... 42
TABLE 6. EXAMPLES OF PLANTS THAT ARE SE ACCUMULATORS OR HYPER
ACCUMULATORS AND ARE PART OF HUMAN FOOD INTAKE (ADAPTED
FROM(ŘEZANKA AND SIGLER 2008)) ...................................................................................... 43
TABLE 7. DAILY SELENIUM INTAKES IN SOME WORLD COUNTRIES. ................................... 44
TABLE 8. OVERVIEW OF ANALYTICAL METHODS FOR DETERMINING SELENIUM IN
BIOLOGICAL MATERIAL. ............................................................................................................. 47
TABLE 9. OVERVIEW OF EXPERIMENTAL DESIGN USED IN THE PROCEDURE, TAKING
INTO CONSIDERATION THE SAMPLING STAGE FOR EACH OF THE DIFFERENT A.
BIOSPORUS TYPES (CYCLE STAGE), ACCORDING TO CROP TYPE AND GROWING
TUNNEL (HOUSE). ........................................................................................................................ 53
TABLE 10. CONDITIONS OF SE METHOD DEFINED ON GFAAS. .............................................. 55
TABLE 11. HEATING PROGRAM OF THE GRAPHITE TUBE ATOMIZER. ................................. 57
TABLE 12. MOISTURE CONTENT OF THREE DIFFERENT TYPES OF A.BISPORUS............ 62
TABLE 13. AVERAGE LEVELS (µGSE/G DRY MUSHROOM) AND RESPECTIVE
STANDARDS DEVIATIONS OF SELENIUM CONCENTRATION IN AGARICUS
BISPORUS OBTAINED IN IRELAND. SELENIUM DISTRIBUTION ACCORDING WITH
CROPPING STAGE IN ONLY TWO TYPES OF MUSHROOMS “BB” AND “CC” DURING 3
DIFFERENT FLUSHES. ................................................................................................................ 64
TABLE 14.SELENIUM DISTRIBUTION SE (µGSE/G DRY MUSHROOM) OF AGARICUS
BISPORUS, AVERAGE LEVELS IN SEQUENCE ACCORDING WITH CROPPING
STAGE, HOUSE WHERE MUSHROOMS GROWN, AND THREE TYPES OF
MUSHROOMS. ............................................................................................................................... 64
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TABLE 15. SIGNIFICANCE VALUES FOR THE MIXED-EFFECT SPLIT-PLOT ANOVA WITH
TWO FACTORS FOR LOG10 |SE| FRESH MUSHROOM. ..................................................... 67
TABLE 16. SELENIUM CONTENT (µG/G SE FRESH MUSHROOM) IN BABY BUTTONS AND
CLOSED CUPS, EXPRESSED VALUES DURING THE FLUSH NUMBER. ........................ 67
TABLE 18. SIGNIFICANCE VALUES FOR THE MIXED-EFFECT SPLIT-PLOT ANOVA WITH
TWO FACTORS FOR THE LOG10 |SE| DRY MUSHROOM ................................................... 70
TABLE 19. SELENIUM CONTENT (µGSE/G MUSHROOM DW) IN BABY BUTTONS,
EXPRESSED VALUES DURING THE FLUSH. ......................................................................... 71
TABLE 21. SIGNIFICANCE VALUES FOR THE MIXED-EFFECT SPLIT-PLOT ANOVA WITH
TWO FACTORS FOR LOG10 |SE| FRESH MUSHROOM ....................................................... 73
TABLE 22. SELENIUM CONCENTRATION (µG/G SE FRESH MUSHROOM) IN
COMMERCIAL A.BISPORUS 3 TYPES OF MUSHROOM IN STUDY. ................................. 73
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INDEX
ACKNOWLEGMENTS ..................................................................................................................... iii
ABSTRACT ...................................................................................................................................... v
RESUMO ........................................................................................................................................vi
ABREVIATIONS LIST ...................................................................................................................... vii
INDEX OF FIGURE .......................................................................................................................... ix
INDEX OF TABLES......................................................................................................................... xiii
INDEX ............................................................................................................................................ xv
PART I: THEORETICAL FRAMEWORK ........................................................................................... 17
1. Introduction............................................................................................................................. 19
1.2. Morphology of Agaricus bisporus .................................................................................... 20
1.3. Mushroom Physiology ...................................................................................................... 22
1.4. Nutritional properties of Mushrooms .............................................................................. 22
1.4.1. Proteins & Amino acids ............................................................................................. 23
1.4.2. Carbohydrates ..................................................................................................... 24
1.4.3. Lipids.................................................................................................................... 24
1.4.4. Vitamins ............................................................................................................... 25
1.4.5. Minerals ............................................................................................................... 26
1.5. Nutritional attributes of A.bisporus ............................................................................ 26
1.6. Agaricus bisporus effects in Human health ................................................................. 27
1.7. Mushroom Production ................................................................................................ 28
1.7.1. Phase 1: Creation of Mushroom Compost ................................................................ 30
1.7.2. Phase 2: Pasteurization of the compost .............................................................. 31
1.7.3. Phase 3: Incubation of the Compost ................................................................... 31
1.7.4. Phase 4: Growing Stage ....................................................................................... 32
1.8. Mushroom industry in Ireland .................................................................................... 36
1.8.1.Location of the Irish Industry ..................................................................................... 37
1.8.2. The mushrooms market ...................................................................................... 38
1.9. Structure and organization of Irish Mushroom Industry ............................................ 39
2. Selenium – General considerations ......................................................................................... 41
2.2. Selenium in the food chain............................................................................................... 43
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2.3. Dietary requirements ....................................................................................................... 44
2.4. Selenium Toxicity ............................................................................................................. 45
3. Review of the analytical methods to quantify Selenium in foods .......................................... 46
4. Objectives ................................................................................................................................ 50
PART II: EXPERIMENTAL DEVELOPMENT ..................................................................................... 51
5. Materials and methods ................................................................................................... 52
5.1. Reagents and materials ............................................................................................... 52
5.2. Mushroom Samples .................................................................................................... 52
5.3. Selenium Determination and Sample Preparation ..................................................... 54
5.5. Standard Preparation and calibration curve ............................................................... 55
5.6. Graphite Furnace Atomic Absorption Spectrometer conditions ................................ 55
5.7. Statistical Analysis ....................................................................................................... 57
6. Results and Discussion ........................................................................................................ 60
6.1. Method development – Sample digestion .................................................................. 60
6.2. Method development – GFAAS calibration................................................................. 61
6.3. Moisture content ........................................................................................................ 62
6.4. Selenium content of Irish Agaricus bisporus ............................................................... 63
6.5. Effect of production and cycle factors on selenium content ...................................... 66
6.5.1. Selenium content in fresh A.bisporus expressed in µg/g fresh mushrooms....... 66
6.5.2. Selenium content in A.bisporus in dry mushrooms ............................................ 70
6.5.3. Effect of growing stage on selenium content of fresh mushrooms .................... 72
6.6. Irish A.bisporus contribution to the Se daily intake .................................................... 74
7. Conclusions ......................................................................................................................... 75
References ................................................................................................................................... 76
Annexes ....................................................................................................................................... 83
PART I: THEORETICAL FRAMEWORK
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1. Introduction
1.1 Mushrooms – Historic Perspective
The origin of the name “mushroom” is derived from the Medieval English
Muscheron, which came from the Old French Mouscheron or Mouseron meaning
“mushroom” which was derived in turn the Old French mousse or mousae, the Teutonic
word for “moss”. Mushroom word refers to something that is able to expand or increase
rapidly (Nonnecke 1989).
Since last 300 million years mushrooms have been part of fungal diversity.
According to the literature, mushrooms were the first food collected in the wild by
prehistoric humans, to use them as a food and also for medicinal purposes(Chang and
Miles 2004).
The first written references about mushrooms date 450 AD and it is found in
Euripides epigram where a poisoning history resulting in death of a local family is
reported
Wild mushrooms as a ritual predate Egyptians, who used mushrooms in their
religious practices and believed that they ensure immortality. Romans considered
mushrooms as the “Food of the Gods”.
Mushroom cultivation did not come into existence until 7th century, when
Chinese cultivated Aricularia auricular, the first mushroom to be cultivated around 1000
AD. These mushrooms were grown outdoors without using any specially prepared
spawns.
(Nonnecke 1989; Chang and Miles 2004) suggest that the most significant
advance in the field occurred in 1650, when French gardeners cultivated accidentally
Agaricus bisporus in Paris, commonly known as champignon or button mushroom. The
first technique for growing domesticated mushrooms was outdoor and use horse
manure as a substrate.
For three centuries mushrooms cultivation has suffered great developments.
The appearing of modern cultivation methods and techniques allowed the growing of
mushrooms indoors using a pure culture spawn containing living mycelium of desired
mushroom species. In 1886 a pure culture mushroom spawn for A.bisporus was first
achieved in United Kingdom, in 1894 in France and in the beginning of 1902 in the
United States(Chang and Miles 2004)
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In the middle of XIX century, United States started the mushrooms cultivation in
New York with spores imported from England were the mushroom commercialization
had already started.
Mushrooms intake in some countries of Europe and US was expand due to an
increase of oriental colonies (Chinese’s, Japanese and Korean). At the moment there
are over than ten thousand known mushroom species, however only approximately two
thousand are considered edible. Of these, 20 were grown commercially.
In Ireland, mushrooms were first grown commercially in the mid 1930’s with exports
to Great Britain beginning in 1947 (Chang and Miles 2004). The most common variety
cultivated is the white button – Agaricus bisporus. The market for mushrooms in the UK
is the largest in Europe at £ 359M.
1.2. Morphology of Agaricus bisporus
Mushrooms belongs to a variety of fungi that can be defined as “a macrofungus
with a distinctive fruiting body which can be either epigeous or hypogeous”. The
macrofungi have fruiting bodies large enough to be seen with the naked eye and to be
picked up by “hand”.
A.bisporus mushrooms consist of three different tissues cap, gills and stalk or stipe,
(Braaksma, van Doorn et al. 1998; Aguirre 2008; Gaston 2010; O'Gorman 2010) as
illustrate in Figure 1.
Cap - is fleshy and hemispherical and as the cap expands it becomes flattened
in order to protect the gills – reproductive tissues. The cap color ranges from
white to cream at first, becoming brownish with age and damage.
Gills – Situated underneath the cap and are the reproductive tissues of the
mushroom and produce millions of spores. In many mushrooms the gills are
covered early in development by a veil and in the mature mushroom the
remains of this veil can be seen as a ring around the stipe. Over time the colour
of the gills change from a pinkish colour to a brown black colour as the spores
mature.
Stipe - is cylindrical and white in colour. It is connected at its base to the
mycelium in the compost. Its function is to lift the cap above the compost in
order for the spores to be released.
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The Mycelium is the vegetative structure stage of the mushroom. It is composed
by the hypae, which consists in microscopic filaments that collectively make up the
mycelium. It forms a felt-like web which ramifies trough the substrate. These filaments
grow only at the tip or at specializes regions and form a system of branching threads
and cordlike strands that branch out throughout the soil or compost. It is quite easy to
see the fungus in its vegetative stage without the help of a hand lens or a microscope.
At certain stage of development of the fungal organism, when the conditions are
favorable, the mycelium gives rise to the mushroom fruit bodies, which are reproductive
structures. The function of the mushrooms is to produce spores and mushroom fruit
bodies are also call sporophores (Chang and Miles 2004).
Table 1 describes the main characteristics of A.bisporus and respective
functions.
Table 1. Resume of A.bisporus morphology characteristics (Adapted from(Chang and Miles 2004)).
A.bisporus Morphology
Structure Function
Cap Covers and protect the gills
Gills Contains hyphae that produce spores
Stalk or Stipe Supports the Cap. Connect to the compost
Spore Cell that develops into new organism
Hyphae Threadlike structure built of fungal cells
Figure 1 Schematic representation of Agaricus bisporus fruiting body on which the different tissues
are indicated (Reproduced from(Mohaček-Grošev, Božac et al. 2001)).
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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1.3. Mushroom Physiology
Mushrooms are heterotrophic organisms they cannot synthesize their own
nutrients, contrary to the plants that can do photosynthesis. Instead, they obtain
their nutrients absorbing soluble inorganic and organic materials from the
environment, from substances like wood logs, manure composts or other organic
composts, so find organic carbon in their substrate is requirement (Beelman,
Royse et al. 2003; Chang and Miles 2004).
This carbon source provides the skeletal carbon for organic compounds and the
energy for the anabolic processes. Other elements necessary for fungal life include:
oxygen, hydrogen, phosphorus, potassium, copper, iron, zinc and vitamins. Three
essential earth elements like, heat, light and water are also essential to fungi for its
role during the growth cycle. (Beelman, Royse et al. 2003) .
1.4. Nutritional properties of Mushrooms
Mushrooms can be grouped into three different categories; (1) edible; (2)
medicinal; and (3) poisonous. Edible mushrooms (mainly the fruiting body) can be
consumed either as flesh (e.g. Agaricus bisporus or usually called button
mushroom) or dried (e.g. Lentinus edodes or shiitake) or preserved in other ways.
Medicinal mushrooms are fungi used not only for culinary purposes but contain
bioactive components (polysaccharides, lypopolysaccharides, glycoproteins and or
bioactive constituents) that have pharmacological properties and consequently
have medicinal application specially used in traditional Chinese medicine (Ruthes,
Rattmann et al. ; Cheung 2010).
The nutritional value of the mushroom originates from their chemical
composition. It should be noted that mushroom composition varies greatly due to
their strains, cultivation techniques (including different substrates), maturity at
harvest and methods of analysis.
In general mushrooms are considered health foods because contain
considerable amounts of protein, dietary fiber, vitamins and minerals and opposite
they are low in fat, calories and energy. Recently mushrooms are reported as a
potential source of nutraceutical substances such as vitamins and minerals (Grube,
Eng et al. 2001; Barros, Cruz et al. 2008; Grangeia, Heleno et al. 2011; Roupas,
Keogh et al. 2012).
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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There are se eral studies a ailable in literature reporting nutrients analysis of
mushrooms (D e and Alvarez 2001; Chang and Miles 2004; Agrahar-Murugkar
and Subbulakshmi 2005; Barros, Baptista et al. 2007; Kalač 2009; Pa el 2009;
Cheung 2010; Aya , Torun et al. 2011; Çağlarirmak 2011; Michael, Bultosa et al.
2011; Costa Orsine, Garbi Novaes et al. 2012; Pereira, Barros et al. 2012; Reis,
Barros et al. 2012).
In these studies different mushrooms specimens were studied by the scientific
community, in searching for substances that may be considered a food or part of a
food and provides medical or health benefits like the prevention and treatment of
some diseases. In these researches authors are also searching for new therapeutic
alternatives, and the results proved their bioactive properties.
1.4.1. Proteins & Amino acids
In general, the crude protein content in edible mushrooms varies significantly
and ranges from 15% to 35% of dry weight (DW), depending on the species,
varieties and stage of development of the fruiting body (Cheung 2010; Michael,
Bultosa et al. 2011).
The proteins of cultivated mushroom contain all the nine essential amino acids i.e.
those which the body cannot synthesize (lysine, methionine, tryptophan, threonine,
valine, leucine, isoleucine, histidine and phenylalanine) (Chang and Miles 2004).
The content of free amino acids in mushrooms is low, only about 1% of dry matter
(Kim, Chung et al. 2009). Their nutritional contribution is thus limited. However, they
participate in the taste of mushrooms. Glutamic acid and alanine were reported as
prevailing free amino acids (D e and Al are 2001).
Mushroom proteins are relatively rich in amino acids threonine (41–95 mg/g
proteinDW), valine (36–89 mg/g protein DW), glutamic acid (130–240 mg/g protein
DW), aspartic acid (91–120 mg/g protein DW %), and arginine (37–140 mg/g
protein DW) but are poor in methionine (1.2–22 mg/g protein DW) and cysteine
(16–19 mg/g protein). It has also been reported that lysine, leucine, isoleucine and
tryptophan are the limiting amino acids in some edible mushrooms (D e and
Alvarez 2001; Cheung 2010).
Mushrooms contain sufficient quantities of B-complex vitamins and vitamin. Protein
levels were comparable to those of cauliflower and whole milk (Çağlarirmak 2011).
Different mushrooms specimens contains different types of free amino acids in
varying amounts (Kim, Chung et al. 2009)
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1.4.2. Carbohydrates
The total carbohydrate content of mushrooms, including digestible and non-
digestible carbohydrate, varies with species and ranges from 35% to 70% DW (D e
and Alvarez 2001; Cheung 2010).
Digestible carbohydrates found in mushrooms include α- trehalose, mannitol
and glucose, are the main representative monosaccharides. Their derivates and
oligosaccharide groups, respectively, usually present in very small amounts (less than
1% DW) and glycogen (5-10% DW). Glycogen is widely consumed, mainly in meat,
and its low intake from mushrooms thus seems to be nutritionally trivial (Kalač 2009).
The major portion of mushroom carbohydrates are non- digestible carbohydrates
include oligosaccharides such as trehalose and non starch polysaccharides (NSPs)
such as chitin, β – glucans and mannans, which corresponding to the major portion of
mushroom carbohydrates (Kalač 2009; Cheung 2010). Water-insoluble structural
polysaccharide such as chitin varies in mushrooms in a range from 80 - 90% DW
(Pavel 2009).
There are limited information on literature about fibre content on mushroom dietary
intake, although, apparently high portion of insoluble fibre seems to be nutritionally
desirable.(Reis, Barros et al. 2012) reported an extraordinarily appreciable level of total
fibre for A.bisporus that gave the highest carbohydrates levels compared with other
mushrooms species. A possible justification for this fact is due to a higher level of non-
fibre carbohydrates such as sugars (Table 2).
1.4.3. Lipids
The constituents of lipids in cultivated and edible mushrooms have been a
interesting area of research since 1980.
According to the extensive literature the macronutrients more specifically total lipids in
edible mushrooms are found in small amounts (Ruthes, Rattmann et al. ; Barros,
Baptista et al. 2007; Kavishree, Hemavathy et al. 2008). In general edible mushrooms
are low in total lipids, crude fat are less than 5% DW (Cheung 2010).
The acids include C12–C20 even-numbered fatty acids and C16–C24 hydroxy fatty
acids, with oleic, linoleic, and palmitic acids predominating. These acids may exist in
their free form or be conjugated to other lipid constituents (Kavishree, Hemavathy et al.
2008).
Even though, linoleic acid is the principal unsaturated fatty acid of mushrooms
lipids, it contributes greatly to the flavor of mushrooms because of its role as the
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precursor to 1-octen-3-ol, which is the main aromatic compound known as fungal
alcohol in most mushrooms. These alcohols, together with two associated C8 Ketones
(1- octen-3-one, 3- octanone), constitute the main volatiles and are considered the
major contributors to the characteristics mushroom flavor (Kavishree, Hemavathy et al.
2008; Pavel 2009; Cheung 2010).
Other fatty acids are present at only low levels and the incidence of trans fatty
acids in mushrooms has not been reported and it’s not expected.
1.4.4. Vitamins
Plenty information about the vitamin contents of wild mushrooms have been
increasingly reported during last decade (Mattila, Lampi et al. 2002; Pavel 2009;
Cheung 2010; Pereira, Barros et al. 2012; Reis, Barros et al. 2012) have data which
demonstrate that edible mushrooms are a rich source of several vitamins including
riboflavin (vitamin B2), thiamine (B1), niacin, biotin and as ascorbic acid (vitamin C).
In accordance with (Cheung 2010) riboflavin content in mushrooms is higher than that
generally found in vegetables, and some varieties of A.bisporus have been reported to
have concentrations as high as those found in eggs and cheese (Mattila, Könkö et al.
2001).
The ergocalciferol (provitamin D) contents of the mushrooms are new and focus
research area of interest. Recently there are data reports that demonstrated that Ergo
is concentrated in mushrooms to levels that make mushrooms by far the best known
dietary source. Ergo is the only known dietary antioxidant that has its own genetically-
coded transporter in humans, considerable interest arisen from researchers world-wide
to investigate its physiological functional and possible nutritional role.
Recent studies have found that cultivated A.bisporus white button mushrooms exposed
to UV light under certain conditions produced vitamin D2 (povitamin D can be
converted into vitamin D in the presence of sunlight) in amounts exceeding the required
adequate intake (Roberts, Teichert et al. 2008; Koyyalamudi, Jeong et al. 2011),
subsequently mushrooms are a rich natural vitamin D source.
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1.4.5. Minerals
Fresh mushrooms have high water content, around 90%.The ash content of
edible mushrooms ranges from 6% to 11% DW and contains a wide variety of minerals.
They are also a good source of minerals. The major mineral constituents are potassium
(K), phosphorus (P), sodium (Na), calcium (Ca), magnesium (Mg) and selenium (Se).
Copper (Cu), zinc (Zn), iron (Fe), manganese (Mn), molybdenum (Mo) and cadmium
(Cd) make up the minor mineral constituents (Chang and Miles 2004; Cheung 2010).
1.5. Nutritional attributes of A.bisporus
White buttons or A.bisporus is the most popular and commercial edible
mushrooms available in world food markets. In order to promote the use of this
specimen of mushroom as source of nutrients and nutraceuticals, some experiments
were performed focused on these commercial specie (Barros, Cruz et al. 2008;
Cheung 2010). This overview focuses on the nutrient and non nutrient compounds in
A.bisporus, as well as the bioactive chemical components or nutraceuticals present in
white buttons (Table 2).
Table 2. Proximate chemical composition (g/100 g) and energetic value (kJ/100 g) of Agaricus
bisporus, values are expressed in a dry weigth (DW) basis. *nd – not detected (Adapted from
(Barros, Cruz et al. 2008))
Nutritional Composition (g/100g) and energetic
value KJ/100g Reference
Crude protein 80.93
(Barros, Cruz et al.
2008)
Crude fat 0.98
Carbohydrate 8.25
Reducing
sugars 1.44
Energy 1550.06
Ash 9.90
Sugar
Composition
Mannitol 19.57
(Barros, Cruz et al.
2008)
Trehalose 0.77
Maltose nd*
Total sugars 20.87
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In general A.bisporus is richer sources of protein and had lower amount of fat.
Carbohydrates were also an abundant nutrient in A.bisporus according with (Barros,
Cruz et al. 2008). A.bisporus has a considerably high concentration of sugars;
otherwise maltose was not found in these commercial mushrooms.
1.6. Agaricus bisporus effects in Human health
The properties and mechanisms of extracts and bioactive compounds from
A.bisporus that have been evaluated in a human population or human cell lines are
outlined in table 3. There are some studies focused on the relationship between
mushroom consumption and breast cancer risk, DNA damage and wound healing
(Roupas, Keogh et al. 2012)
Table 3. Properties and mechanisms of bioactive compounds and A.bisporus extracts evaluated in
animal models or animal cell lines (Adapted from (Roupas, Keogh et al. 2012))
Effect/disease
state
Bioactive or
extract Mechanism (in vitro/in vivo) Reference
Anti-cancer
(colorectal) Lectin
Inhibit the proliferation of HT29
human colonic cells (in vitro -in
human cells)
(Yu, Fernig et
al. 1993)
Anti-cancer
(breast)
Aqueous
extracts
Suppress aromatase activity and
proliferation of MCF-7aro cells-
hence suggesting a reduction in
estrogen production (breast cancer
cell lines)
(Grube, Eng et
al. 2001)
DNA damage Heat-labile
protein
Protect Raji cells (human
lymphoma cell line) against
H2O2 -induced oxidative
damage to cellular DNA
(in vitro)
(Shi, Benzie et
al. 2002)
Wound healing
Unspecified
bioactive/
extract(s)
Dose-dependent inhibition
of proliferation and lattice
contraction in an in vitro
model of wound healing
(Batterbury,
Tebbs et al.
2002)
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(human ocular firoblasts in
monolayers and in 3-D
collagen lattices)
A. bisporus is the most commonly cultivated and consumed edible mushroom.
However, there are few reports attributing medicinal properties to this fungus although
quinoid compounds obtained from this mushroom suppressed the propagation of
mouse ascites tumour, and a lectin from this species also reversibly inhibited the
proliferation of human colon carcinoma cells. Recent studies have shown that cold
water extracts of A. bisporus fruit bodies prevented H2O2 - induced oxidative damage to
cellular DNA but the nature of the protective mechanism was not identified (Shi, Benzie
et al. 2002).
Recently a research data confirmed that A.bisporus is a potent but reversible
inhibitor of ocular fibroblast proliferation and collagen lattice contraction yet lacks
citotoxicity. A.bisporus may therefore be suitable agent for modulating wound healing in
the subconjunctival space after glaucoma surgery(Batterbury, Tebbs et al. 2002)
Other study showed that A.bisporus lectin causes dose-dependent inhibition of
proliferation of HT29 human colorectal carcinoma cells, human breast cancer MCF-7
cells and rat mammary fibroblast Rama-27 cells (Yu, Fernig et al. 1993).
Mushrooms and mushroom bioactive components have been reported to have
numerous of positive health benefit effects, mainly on the basis of in vitro and in vivo
animal trials.
1.7. Mushroom Production
Due to a large consumption of mushrooms in last decades at the same time
were being noticed a gradual development of mushrooms production.
Mushroom growing is one of the most unusual stories in agriculture. In 1959 in
Denmark was developed the use of plastic bags for mushroom growing and spread to
France and Germany (Teagasc 1994). The technology underpinning Irish adaptation of
this growing system was developed at The Irish Agriculture and Food Development
Authority research center - Tegasc. The basis of the expansion of the Irish mushroom
industry was started with the system of growing in plastic bags (Figure 2) in the 1980s.
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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Figure 2. A. bisporus growing in a polyethylene bag (Reproduced from (O'Gorman 2010)).
The large volume of air over the cropping surface and the deep layer of
compost in each bag, produced mushrooms that were of better quality than those
produced from a multilayered system. This quality advantage played a critical role in
enabling the Irish industry to gain a significant share of the UK fresh mushroom market
(Teagasc 1994).
Bag system requires a high manual labor input and this is one of disadvantages
of this first system implemented in mushroom farming.
In order to industrialize mushrooms growing, in the last ten years, mushroom
industry has done some modifications, Irish mushrooms companies choose to replace
plastic bags for more mechanized shelf system (shelves), and nowadays more
automatyzed system and mechanism is being used.
Good mushroom substrate (compost) and the right environmental conditions
are the two essential requirements for mushroom growing. As a compost quality is
largely outside the control of mushroom growers, their main contribution to final product
quality is crop management. This involves controlling temperature, relative humidity,
watering, and ventilation and CO2 levels. Actually modern mushroom houses are
equipped with computerized environmental control systems for this purpose (Teagasc
1994)
During the crop cycle, mushrooms are harvested in a rhythmic pattern of breaks
or flushes that occur at approximately seven day intervals. After two flushes, production
declines rapidly and a grower must decide to terminate the crop and start anew or face
dwindling harvest of mushrooms from each successive flush (Teagasc 1994; Aguirre
2008; Gaston 2010).
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Follows a summary of how the most popular varieties are cultivated, mainly
based on a visit processing to one of the hugest mushrooms companies in Ireland –
Monghan Mushrooms Lda.
In accordance with Tegasc and with Monaghan Mushrooms Lda, there are four
steps involved in mushroom production (Monaghan Mushrooms. (May 2012; Tegasc.
(May 2012).
Figure 3. Representative scheme of Mushroom growing cycle (Adapted from (Monaghan Mushrooms. (May 2012)
1.7.1. Phase 1: Creation of Mushroom Compost
The sequence used to produce this specific substrate for the mushroom is called
composting or compost substrate preparation and is divided into three stages, Phase I
Phase II, and Phase III. Each stage has distinct goals or objectives. It is grower’s
responsibility to provide the necessary ingredients and environmental conditions for the
chemical and biological processes required to complete these goals. The management
of starting ingredients and the proper conditions for composting make growing
mushrooms so demanding.
Mushroom compost is made to meet the very specific requirements for the growth
and fruiting of mushrooms.
Bales of wheat straw are mixed with recycled poultry material, water and other
organic material. When mixed, the material immediately gets put into large chambers,
called aerated bunkers. During this phase the substrate reaches temperatures of 80ºC.
After 13 days the finished substrate is ready to be pasteurized and conditioned.
1. Creation of Mushroom Compost
2.Pasteurization of the compost
3. Incubation of the Compost
4. Growing Stage
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Figure 4. The initial phase of creation of compost. This aerated substrate preparation system has
piped concrete floor under the substrate that forces air through the substrate to maintain aerobic
conditions during the composting process (Reproduced from (Beyer and Extension 1997)).
1.7.2. Phase 2: Pasteurization of the compost
The substrate is then delivered to the pasteurization tunnels to eliminate the bad
microbes such as insects, other fungi, and bacteria. This is not a complete sterilization
but a selective killing of pests that will compete for food or directly attack the
mushroom.At the same time, this process minimizes the loss of good microbes.
Pasteurization and conditioning of the substrate takes approximately 6 days. The
climate controlled “tunnel” heats the substrate to 58 ºC for pasteuri ation and then
conditions it at 48 ºC.
Figure 5.Self-propelled compost turner moving though a compost rick or pile (Reproduced from
(Beyer and Extension 1997)).
1.7.3. Phase 3: Incubation of the Compost
At the end of the conditioning process the substrate is then cooled down to 26 ºC.
The substrate is then transferred to an incubation tunnel. During this transferring
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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process spawn is added to the substrate. Spawn is usually made with rye or wheat
grain that has been sterilized and inoculated with mushroom tissue (mycelium).
This incubation process takes 14-17 days. During this time the white fuzzy mycelium
grows throughout the substrate.
After the 14-17 day incubation period, the substrate mixture is loaded into specially
designed lorries for transport to the growing rooms.
Figure 6. Representation of the last phase of composting. (a) handful of composted substrate
showing white – flecking (“firefang”)microbial growth. (b)Spawn grains used to seed the compost
with mushroom mycelia. Spawn is cooked, sterilized,grain colled, and inoculated with mushroom
mycelia (Reproduced from (Beyer and Extension 1997))
1.7.4. Phase 4: Growing Stage
As the mushroom substrate is filled into the growing rooms a layer of peat is
applied to the surface of the compost. The layer is called the casing layer and is
essential for the formation of the mushrooms. Over a 3-4 day period, the mushroom
tissue grows throughout the substrate and up through the casing layer.
Casing: the only method of forcing mushroom mycelia to change from the
vegetative phase to a reproductive state is to apply a cover of a suitable
material – called casing layer – on the surface of the spawned compost.
Mushroom casing is a layer of organic material (usually neutralized peat)
which is applied to the surface of the spawn-run compost. The function of a
casing layer is to trigger the mushrooms to switch from vegetative growth to
reproductive or fruiting growth. Temperature, relative humidity, CO2 and
watering must be controlled between the precise day of casing and crop
initiation (Teagasc 1994).
(a) (b)
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Figure 7. Spawn growth in the casing and its thicker rhizomorph growth (Reproduced from (Beyer
and Extension 1997))
The environment is then altered to stimulate an autumn day, which stimulates
the formation of mushrooms. As a result, tiny mushroom heads (pins) begin to appear.
During the next two weeks the levels of moisture, temperature, humidity, carbon
dioxide and air movement are carefully monitored. Such as introducing fresh air,
decreasing the temperature, reducing CO2 level and maintaining the relative humidity,
will cause breaking and subsequent pinning. A few days after the beginning of the
breaking, the pins will be visible as white clusters, which are formed by the fusion of
mycelia strands
The pins eventually grow into mushrooms and start the harvest (Figure 8).
Harvest: the first flush of mushrooms occurs 2-3 weeks after casing and
lasts for 3-5 days. The crop develops in four flushes in weekly cycles. The
first two flushes usually provide 70% of the total yield. Harvesting must
occur when the cap is at its maximum size before the veil has stretched and
opened, exposing the gills. Care must be taken not to remove excessive
casing, which would remove the pinheads required to form later flushes.
Mushrooms are harvested by hand and picked at time before the cap
becomes soft,indicating the mushrooms room is past prime fresh-quality
potential. Harvesting rates depend mainly on the amount of crop on the
beds and size of the mushrooms. Growers harvest just three to four breaks
per crop – a shorter harvesting time allows more crops to be produced in a
year and helps to prevent disease and insect problems.
Pinning : Mushroom initials develop after rhizomorphs have formed in the
casing. The initials are extremely small but can be seen as clumps on a
rhizomorph. As these structures grow and expand, they are called
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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primordial or pins (Figure 8(a)). Mushroom pins continue to grow larger
through a prebutton stage and ultimately enlarge to mature mushrooms.
Mushroom harvesting begins 15–21 days after casing, which is normally
10–12 day after flushing and 7–8 weeks after composting started (Beyer
and Extension 1997).
Figure 8. The developmental stages of the Agaricus bisporus fruiting process. (a) mycelium;
(b)initials-clumping; (c)pin-primordia; (d)pea-sized pin; (e) pre-white button (Reproduced from
(Beyer and Extension 1997).
(a) (b)
(c) (d)
(e)
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The mushrooms are picked by hand to maintain the highest possible quality. All
mushrooms are cooled quickly after harvesting and transported in specifically designed
refrigerated trucks.
When the mushroom cycle is complete the compost is killed off using and
compost temperature is held for 6-9 hours at around 70ºC, in order to reduce the
chances of contaminating the subsequent crops.
(Leonard and Staff 1999) describe five main characteristics which characterize
the ideal grade mushrooms Table 2.
Table 4. Optimum characteristic of prime grade mushroom (Adapted from(Leonard and Staff 1999)).
Characteristics Description
Maturity Caps completely closed with no sign of the veil
Appearance
Caps clear white in color with no signs of damage or discoloration
any kind. Stipe clear white in color with no damage or splitting a right
angle cut at the end
Size
Cap size is specified by the market outlet and varies somewhat
company to company. And the stipe length is also specified by
market outlet.
Shape Cap must be firm and rounded and not misshapen. Stipe must also
firm and rounded with no hollow steams.
Peat Traces of peat are not allowed.
There are two systems of growing crops in mushroom houses:
Single-layer bag-growing in tunnels (Figure 4.a). It’s a simple and effective
method where specialized companies make and supply the mushroom
producers with ready-to-use compost bags that contain mushroom spawn
mixed through it. The large volume of air over the cropping surface and the
deep layer of compost in each plastic bag favor the growth of good quality
mushrooms. The easy disposal of cropping remains allows for efficient
environmental control and better.
Multi-layer systems on shelves (Figure 4.b), which can double, triple or
quadruple the output of a tunnel . This method allows mechanized compost
filling/emptying and facilitates the use of automated harvesting equipment,
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
36
which results in a reduction in labour input. (This is the method used in
Monaghan Mushrooms Lda)
Figure 9. Mushroom growing systems (a) Single-layer bag growing. (b) Multi-layer structure for growing shelves
(Reproduced from (Marshall 2009)).
In accordance with (Tegasc. (May 2012) the majority of Irish mushrooms farms
actually are using shelf farms with multi-layer structure for growing shelves (Figure
4.b), including Monaghan Mushrooms Lda.
1.8. Mushroom industry in Ireland
The development of mushroom industry in Ireland has been one of the most
spectacular successes of Irish horticulture in recent years.
Mushroom production has expanded steadily over the past decade and there are
approximately 580 growers throughout the country. Ireland is now exporting over
35,000 tones of fresh mushrooms, while consumption on the domestic market is
approaching 10,000 tonnes. Total production is around 50,000 tonnes per annum with
70% exported (Tegasc 2000).
The mushroom industry expanded dramatically during the 1980s and 1990s with
the introduction of a new concept of growing called the ‘satellite’ system. The satellite
system was invented in Ireland and is quite simple. Compost companies would sell
compost to an associated group of growers, and then buy the mushroom crop back
from the growers. Further, marketing of the mushrooms was handled by the sales
organization of the compost company. This resulted in a very efficient production and
(a) (b)
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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marketing system, with growers having a secure source of compost and a guaranteed
market for their mushrooms (Mushroom Business. (June 2012). The fact that marketing
of the mushrooms is carried out largely by the compost manufacturers, means that the
marketing is far more organized than for any other Irish horticultural product (Tegasc
2000).
The Irish climate offers great conditions to mushrooms growers, in winter time with
temperatures of around 5ºC and in the summer time 15ºC. This kind of favorable
weather erases the requirement for investments in climate installations for extreme
conditions. The growing areas can be kept simple with some heating and computerized
systems. The weather presents also great conditions for compost production.
Subsequently Ireland’s abundant natural resources, in all aspects, contribute as a
benefits to mushroom production, and ensure that the country will continue to play a
major role in international growing (Mushroom Business. (June 2012).
Mushrooms have been a major success story in diversification, providing income
and employment on many small farms. It’s an ideal complementary enterprise on farms
where there is available labor. Full time employment in the mushroom industry in 1998
was 1,400 with another 3,500 part time jobs, underlining this industry’s importance in
providing employment in rural areas (Tegasc 2000)
Until 2004 Ireland was the third one in the world ranking of fresh-mushroom
exports, after China and the Netherlands. But, in recent years there has been a vast
incensement in Poland’s mushroom production, and nowadays they are the biggest
European’s producers and mushrooms exporters.
After that, and as expected, there’s a competition between Netherlands and Poland,
which reproduce a negative impact on established mushroom industries throughout
Europe, with the numbers of farms declining in most mushroom producing countries.
1.8.1.Location of the Irish Industry
The data on mushroom compost usage and number of mushroom farms show that
the industry is widely distributed throughout the country but with a great concentration
in Monaghan (24% of production) followed by Cavan (11%), Roscommon (9%), Mayo
(8%) and Donegal (7%). Other important mushroom producing counties are Wexford
(6%), Kildare (5%), Meath (4%), Louth (4%) and Galway (4%) (Tegasc 2000).
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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Figure 10. Main growing regions in Ireland (Reproduced from (Bord Bia Irish Food Board. (August
2012))
The majority of mushrooms are grown in the border countries of Ulster, the
North West, the Midlands and the South East. The highly controlled growing
environment (within the polythene tunnels) and the use of pre-manufactured compost
allow mushrooms to be grown anywhere in Ireland, irrespective of soils or climate. As
such the mushrooms sector has strong links into rural farming communities seeking an
alternative enterprise. In Republic the main and major counties involved in mushroom
production is Monagha (Bord Bia Irish Food Board. (August 2012).
1.8.2. The mushrooms market
The main outlet for Irish mushrooms is in the UK. A reputation for quality,
consistency and timely delivery has been gained there, mainly through the central
marketing structures (Tegasc 2000; Tegasc 2000; Tegasc. (May 2012).
The production of quality mushrooms requires a high level of competence and
skill. Mushrooms for the fresh market must be very carefully picked. The market outlet
determines the type and size of the packages that growers may use (Tegasc 2000;
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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Monaghan Mushrooms. (May 2012). The major retail multiples have the dominant
market share of fresh produce in Ireland, which is estimated at 75%-80% (Bord Bia
Irish Food Board. (August 2012).
For example Monaghan mushrooms, one of the biggest mushroom producers in
Ireland have as a clients the national and international retailers leaders as well as Aldi,
Lidl, and Tesco supermarkets.
Despite the difficulties faced over the past few years, growers are investing in
order to improve the growing system, which will allow the Irish industry to maintain a
strong position in the European export market place.
1.9. Structure and organization of Irish Mushroom Industry
The mushroom sector is a modern, quality focused and exports orientated
sector. Traditionally in Ireland, mushrooms are relatively high value produce items
grown for the premium fresh market i.e. the domestic and export market.
There are seven main key players in structure of mushroom industry, they are:
growers; spawn manufacturer; compost companies; wholesalers; marketing
companies/facilitators; prepares and processors. Growers sell mushrooms to the
market, spawn manufacturer make a research work to developing strains mushrooms;
compost companies buy in and mix raw materials with mushroom inoculums.
Wholesalers are responsible for distribution/sale of mushrooms, and marketing
companies act as consolidators for one or more retail multiples. Preparers include
catering/retail pack whole or sliced/diced mushrooms, and fresh salads an the last key
player, processors purchase mushrooms in bulk for use in further processing as a
component of other value added foods (e.g. soups, ready meals, pizza) (Organigram 1)
(Bord Bia Irish Food Board. (August 2012).
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Diagram 1. Structure of mushroom industry. Mushroom supply chain. (Adapted from(Bord Bia Irish
Food Board. (August 2012)).
Spawn Production
Compost Companies
Mushroom Growers
Prepared Sliced,dliced Marketing Company or
Facilitator
Processed Mushrooms
Canned,frozen,dried,etc.
Mushroom
Processes/prepared
product imports
Retail; Multiples;
Sympols; Independent
Food servisse/Catering Wholesalers
Exports Consumer
Domestic
2. Selenium – General considerations
Selenium is an essential trace mineral for living organisms. Selenium is an
essential nutrient of indispensable importance to human biology, subsequently to
human health (Margaret P 2000; Costa-Silva, Marques et al. 2011; Thiry, Ruttens et al.
2012).
Selenium is a chemical element available on periodic table with symbol Se and
atomic number 34; it is included on nonmetal group.
For (Margaret P 2000; Thiry, Ruttens et al. 2012) Se is a micronutrient incorporated
in active centre of selenoproteins, where some of them have crucial enzymatic
functions. Se is a part of the active centre of glutathione peroxidase (GPx) an enzyme
whose role is to protect tissues against oxidative stress by catalyzing reduction of
peroxidases, responsible of various cellular damages (Zeng and Combs Jr 2008).
In last decades were recogni ed se eral proteins and en ymes as “selenoproteins”, in
which one of them were indentified Se exclusively as selenocysteine (SeCys) residue.
These enzymes are selenium-dependent, generally with selenocysteine at the active
site (Margaret P 2000; Helinä 2005; Brigelius-Flohé 2006; Rayman, Infante et al. 2008;
Thiry, Ruttens et al. 2012).
Selenoprotein P (SeP) is the most abundant selenoprotein in plasma and
probably acts as a Se transporter between the liver and other organs such as the brain,
and kidneys (Rayman, Infante et al. 2008).
Nowadays one of the most recognized functions of Se as a trace element for humans
is the health effects particularly associated of specific diseases such as the relation to
the immune response and cancer prevention. There have been reported , in several
epidemiological studies (Margaret P 2000; Helinä 2005; Rayman, Infante et al. 2008;
Thiry, Ruttens et al. 2012) (Zeng and Combs Jr 2008) that less – over selenium
deficiency can promote some diseases directly related with immune function; viral
infections; reproduction; thyroid function; cardiovascular diseases; and even cancer.
Plus, there were been illustrated by the occurrence of specific diseases in areas with
low environment Se levels, Keshan disease is a well known example of an endemic
cardiomyopathy that has been observed in children, adolescent s and pregnant women
in the Keshan region of China, a place where Se levels in soil and food are extremely
low (Thiry, Ruttens et al. 2012)
The major forms of selenium in diet are highly available. Selenium bioavailability
varies according to geographic location.
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2.1. Bioavailability of Selenium
Bioavailability of a nutrient, is usually defined as that fraction of ingested nutrient
that is used for regular physiological functions; absorption and retention of the nutrient
are taken as indirect measures of bioavailability as these are measurable though they
cannot address functional bioavailability which is that most likely to be relevant to
health (Rayman, Infante et al. 2008).
Selenium portion in the en ironment is ery low. In the Earth’s crust, Se is present
in small concentration of 0.05 – 0.09 mg/kg (Table 5). In compounds, selenium is
present as Se 2- , Se 2+, Se 4+ and Se 6+. In the environment, usually selenium is present
in elemental form or in the form of selenide (Se2-), selenate (SeO42-), or selenite
(SeO32) (Ře anka and Sigler 2008).
The Se cycle begins and ends with soil, and the chemical forms (dissolved in soil
solution, adsorbed on the oxide surfaces, fixed in the mineral lattice) and
concentrations of Se in soil determine its bioavailability and thus the need for dietary
supplementation (Helinä 2005). Se form, can be expected to occur under high oxidative
conditions, subsequently, in the soils the amounts of the various oxidation state
species depend strongly on the redox-potential conditions, with the lower oxidation
states predominating in anaerobic conditions and acidic soils, while the higher
oxidation states are favored in alkaline and aerobic conditions (Finley 2006; Ře anka
and Sigler 2008).
Table 5 Overview of general Selenium amounts in Environment (adapted from (Řezanka and Sigler 2008))
Environmental Elements Se Concentration
Earth’s crust 0.05 – 0.09 mg/Kg
Water 0.45 µg/Kg
Stream Water 0,2 µg/Kg
Organic forms of Se (wheat Se, SeMet and high-Se-yeast) were found to be
more bioavailable than selenate and selenite in that they were more effective in raising
blood Se concentrations (suggesting better absorption and retention), though all forms
were able to increase selenoenzyme (glutathione peroxidase) activity (Rayman, Infante
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et al. 2008). Overall, absorption of all forms of Se is relatively high (70% to 95%), but
varies according to the source and the Se status of the subject (Finley 2006).
2.2. Selenium in the food chain
Se content in food and beverages varies in different parts of the world from country
to country, because its level in soil changes with native substrate, climatic conditions
and vegetation cover .The Se content of animal products reflects the Se levels in their
dietary intake (Barclay, MacPherson et al. 1995; Sirichakwal, Puwastien et al. 2005;
Navarro-Alarcon and Cabrera-Vique 2008).
In European countries, crab liver, other shellfish, and fish are moderately good
sources of Selenium. In North America, wheat is a good source of Se, and in Latin
America, more specifically in Brazil, nuts accumulate significantly amounts of Se
(Margaret P 2000) (Table 3). A considerable range in selenium content of soya
products was found in recent study about the relevance of this nutrient in Thai food
(Sirichakwal, Puwastien et al. 2005)
Most plants do not have the ability to accumulate large amounts of Se
(concentrations rarely exceed 100 μg/g, dry weight). However, various plant species
such as garlic, Indian mustard, and some mushrooms species have been recognized
as Se accumulators. They have the ability to take up large amounts of Se (1000 mg
Se/kg) without exhibiting any negative effects.
Table 6. Examples of plants that are Se accumulators or hyper accumulators and are part of human food intake (adapted from(Řezanka and Sigler 2008))
Plant / Se Accumulators Se concentration
(mg/Kg)
Accumulators
Brazil nuts 2.0 – 35 and more
Brussels sprouts 0.03 – 7.0
Mushrooms 0.1 – 20
Wheat 0.1 – 15
Hyperaccumulators
Garlic >1200
Broccoli <300
Ramp >500
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2.3. Dietary requirements
Human dietary intakes of Selenium also range from high to low according with
geography, which decide the Se bioavailability.
In Europe, Se intake ranges approximately from 28µg to 70 µg Se per day
(Margaret P 2000; Navarro-Alarcon and Cabrera-Vique 2008)
The UK Reference Nutrient Intake (NRI) of Selenium is generally below the
reference nutrient intakes (30- 40 µg/day) (Barclay, MacPherson et al. 1995; Margaret
P 2000).
Intake for Selenium in the USA is 55 μg/day for adult men and women (Directorate
2000), and in Canada 50-200 µg/day (Clark, Cantor et al. 1991; Mistry, Broughton
Pipkin et al. 2012). Dietary selenium intake in most parts of Europe is considerably
lower than in the United States, mainly because of the European soils that provide a
poorer source of selenium (Mistry, Broughton Pipkin et al. 2012)
A World Health Organization and Food and Agriculture Organization of the United
States (WHO/FAO) expert group, recommended an intake level of only 40 µg per day
for men and 30 µg for women in China (WHO 1996). Assessments of requirements,
adequacy, and intakes of selenium have been reviewed previously in detail and
summarized in Table 7.
Table 7. Daily selenium intakes in some world countries.
Country Se Intake (µg
per day) Information source
Europe 28 - 70 (Margaret P 2000; Navarro-Alarcon and Cabrera-Vique
2008; Thiry, Ruttens et al. 2012)
UK 30 - 40 (Barclay, MacPherson et al. 1995; Margaret P 2000;
Mistry, Broughton Pipkin et al. 2012)
USA 55 - 220 (Clark, Cantor et al. 1991; Mistry, Broughton Pipkin et al.
2012)
Canada 50 – 200 (Mistry, Broughton Pipkin et al. 2012)
China 30 - 40 (WHO 1996)
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2.4. Selenium Toxicity
Until 1950s Se had been considered merely as an environmental toxicant. The
findings in the 1980s that Se excess caused death of aquatic birds, malformation of
bird embryos and poisoning of fish in California gave rise further environmental
concern regarding this essential element (Helinä 2005).
The toxicity of Se and the mechanisms by which this element exerts its toxic effects
depend on its form, though there are few species-specific data on the toxicity of Se in
humans and none relating to dose or safe upper limits of particular species.(Rayman,
Infante et al. 2008)
In 1996 were carried out some researches which suited the interaction of Se with
toxic metals in the food supply. Were recognized evidences that Selenium seems to
reduce the toxicity of several metals by forming inert metal selenide complexes. For
example, Mercury in marine foods is found combined with selenium, which may
protect against mercury toxicity, subsequently this interaction reduce the bioavailability
of Selenium from such foods (Margaret P 2000; Rayman, Infante et al. 2008)
Other suggested mechanisms of Se toxicity include inhibition of Se methylation, the
major detoxification pathway for Se, allowing the accumulation of hepato-toxic
selenides, notably H2Se. For instance, in mice, high doses of SeCys have been shown
to cause hepatic toxicity by depressing Se methylation through the inactivation of
methionine adenosyltransferase, the enzyme responsible for S-adenosyl methionine
synthetized (Directorate 2000; Mistry, Broughton Pipkin et al. 2012)
Chronic toxicity of selenium in humans results in selenosis, a condition
characterized by brittleness or loss of hair and nails, gastrointestinal problems, rashes,
garlic breath odor, and nervous system abnormalities (Yang, Wang et al. 1983; Mistry,
Broughton Pipkin et al. 2012)
In China, it has been reported that selenosis occurs with increased frequency in
people who consumed selenium at levels above 850 µg/d. The Institute of Medicine
(United States) has set a tolerable upper intake level for selenium at 400 µg/d for adults
to prevent the risk of developing selenosis. The European Commission and the World
Health Organization have proposed the lower daily upper limit of 300 µg/d for adults
(Barclay, MacPherson et al. 1995; Directorate 2000; Margaret P 2000; Mistry,
Broughton Pipkin et al. 2012).
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3. Review of the analytical methods to quantify
Selenium in foods
The determination of selenium is of considerable interest because it would
appear to be an essential trace element but is also toxic at relatively low levels.
Methods for its determination in biological materials and water are critically evaluated
with particular attention given to methods which are widely used in routine analysis
(Campbell 1984)
The essential role of Se in physiology has encouraged the development of
analytical methods for its quantification at trace levels, and a variety of analytical
methods can be used to determine trace concentrations (ng/g) of selenium in biological
tissues. These include fluorometry, neutron activation analysis (NAA), atomic
absorption spectroscopy (AAS), inductively coupled plasma-atomic emission
spectroscopy (ICP-AES), inductively coupled plasma-mass spectrometry (ICP-MS), or
either via hydride generation (HG-AAS), gas chromatography (GC), spectrophotometry,
x-ray fluorescence analysis, and others (Dauchy, Potin-Gautier et al. 1994). The
analytical methods used to quantify selenium in biological and environmental samples
are summarized below on (Table 8).
Although an extensive range of analytical methods is available for selenium, two
methods in particular, molecular fluorescence and atomic absorption spectroscopy,
have adequate sensitivity, require only readily available laboratory apparatus and are
quite suitable for routine survey work (Campbell 1984).
The fluorimetric method is widely accepted as a technique for the determination
of selenium in foods and in biological material, and it is considered the method of
longest standing. Fluorimetry has been applied to several longitudinal studies
investigating the selenium status of milk (Foster and Sumar 1995) and also in
mushrooms samples (Costa-Silva, Marques et al. 2011). Following wet digestion, the
selenium is converted to Se (IV) by boiling with hydrochloric acid, and determined by
measurement of fluorescence formed on the reaction. The sensitivity is acceptable per
sample although the amount of manipulation required in the manual method is
considerable (Campbell 1984; Foster and Sumar 1995).
The determination of selenium by atomic absorption spectroscopy has been
reviewed by some authors (Campbell 1984; Jacobson and Lockitch 1988; Dauchy,
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Potin-Gautier et al. 1994), with application of two types of techniques AAS and graphite
furnace atomic absorption spectrometry (GFAAS).
Classical flame AAS techniques apparently do not have adequate low detection
limits for selenium to be useful for determining its presence in biological samples. In the
other hand GFAAS offers high sensitivity, and it is routinely used method for
determination of numerous metals in foods and other matrices. Organic materials are
then destroyed by high temperature in the furnace prior to atomization of the sample at
extremely high temperatures (e.g., 2700ºC). One advantage of GFAAS techniques is
that material in the graphite sample cell can be chemically treated in situ to reduce
chemical interference (Beaty 1978).
Table 8. Overview of analytical methods for determining selenium in biological material.
Sample
Matrix Preparation method Analytical method Reference
Food
Acidic digestion with HNO3 AA gaseous hydride Epa Methods
7741A
Samples dilutions in HNO3 (1+1
v/v) GFAAS
(Oliveira, Neto
et al. 2005)
Sample digestion with HNO3 and
adjusted ph with ammonia
solution using a mixed solution
0.01 sulphuric acid
Fluorimetric
(Sirichakwal,
Puwastien et al.
2005)
Samples digestion with nitric
acid,perchloric,and sulfuric acid HG - AAS
(Barclay,
MacPherson et
al. 1995)
Samples digestions in a mixture
of sulfuric, perchloric and nitric
acids
Fluorimetric (Yang, Wang et
al. 1983)
Dried samples and incorporated
into modified torula yeast
Fluorimetric using
diaminonapthalene
(Spallholz and
Shi 1994)
Samples digestion with nitric
acid 50% (v/v) HG-AAS
(Sigrist, Brusa
et al. 2012)
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Sample digestion with nitric acid ICP-MS (Choi, Kim et al.
2009)
Predigestion of samples using a
mixture of nitric acid, sulfuric,
and perchloric acids
HG-AAS
(Klapec, Mandić
et al. 2004)
Reported detection limits vary not only with the technique but also with the
parameters used in that technique. It is therefore difficult to generalize. With
conventional flame atomic absorption spectroscopy but using a nitrogen-hydrogen air
entrained flame the detection limit for selenium is about 2-5 µg/cm3, with hydride
generation the detection limit with hydrogen flame atomization is lowered to about 2 ng/
cm3 and practical lower limits of around 5 ng/g of sample are readily attained (Campbell
1984).
Sample stability is a prerequisite for accurate and meaningful chemical
speciation. The conditions used for sample storage and the method of sample
preparation must prevent or minimize changes which affect the integrity of the
selenium-containing species (Patching and Gardiner 1999).
Graphite furnace atomic absorption spectrometry (GFAAS) has been applied to
the analysis of food and water samples for the direct determination of numerous trace
metal elements (Yan-zhong, Mei et al. 1997).
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4. Objectives
The general objectives of this study was to develop an analytical method that
allows the determination of selenium in mushroom samples using atomic absorption
coupled to a graphite furnace - Graphite furnace atomic absorption (GFASS).
The aim of this work was to analyze the quantity of selenium among three
different types of Agaricus bisporus, edible mushrooms, commercially available and
also to analyze the effect of growing conditions on them.
In this sense, for development of GFAAS method for quantification of selenium
in Irish mushroom samples numerous studies and preliminary experiments were
developed that led to the most appropriates and satisfactory results:
i) The study of different conditions of GFAAS with Selenium bulk solutions at
different concentrations in order to obtain the best selenium peak resolution
- calibration curve, and identification of Selenium;
ii) The decomposition of mushroom samples was an important part of
combined analytical methods, which justified study mushroom digestion
conditions taking into consideration the acid used in the digestion process,
dried times and temperatures, and also used concentration of mushroom
solutions;
iii) The optimization of the mushroom digestion procedure;
iv) After the successful development of method, the application of GFAAS
method to real Agaricus bisporus samples.
Following the selenium concentrations in A.bisporus by GFAAS, the next step
was analyze statistically and verify if there are any significant differences between
factors in study: crop/house/flush, between 3 types of A.bipsorus.
The main objective was to report the distribution of selenium in a group of
common, edible mushrooms collected in Monaghan Mushrooms Company.
PART II: EXPERIMENTAL DEVELOPMENT
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5. Materials and methods
5.1. Reagents and materials
Selenium atomic absorption standard were obtained from Sigma-Aldrich, and Nitric
acid (69%) were obtained from Merk. Deionized, water were obtained from SGS water
purification system and ultra-high water was prepared using a Milli-Q Academic
System. Conventional lab materials were used and the entire procedure of mushrooms
digestions were done in fume hoods equipped with automatic extractors. All the glass
material used were previously rinsed with nitric acid.
5.2. Mushroom Samples
The cultivated and edible mushrooms Agaricus bisporus, were directly collected
from the same producer Monaghan Mushrooms Ltd, Ireland, who kindly supplied our
samples, and each 44 samples were transported in individual plastic mushrooms
punnets wrapped with a plastic film. The samples were stored on each punnet at DIT in
a lab freezer at - 4ºC. A list of all 44 Irish samples studied is presented in Table 9. In
these 44 samples were three different types of A.bisporus: Baby Buttons “BB”; Closed
Cups “CC” and Flats “F”. “BB” these are extra small mushrooms, with membranes
closed, only just forming. Steam length does not exceed 2cm (¾ inch), cap diameter
2.5 to 6 cm (1 to ½ inches). CC is tightly with no gills showing, mushrooms with
membranes well developed or just opening, with cap retaining a pronounced cap
shape. Stem length not to exceed 2.5 cm (1 inch) from the apex. Cap diameter 2.5 to 7
cm (1 to 2¾ inch). “F” a fully opened mushroom, usually medium to large in si e.
Mushrooms that ha e ad anced beyond the cap stage, the cap forming the letter ‘T’
with the stipe. Cap diameter 2.5 to 7 cm (1 to 3½ inch) and stem length not to exceed
2.5 or 3 cm, according to the class. All these standards follows the issued a small
booklet called “International Standards for Edible Fungi” (Codex Alimentarius
commission No.38) in 1970 by The Food and Agricultural Organization of the United
Nations (FAO).
There are also three distinct houses (tunnels) where mushrooms were growing up in a
farm. Modern mushroom houses are equipped with computerized environmental
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control systems for this purpose. Two different agricultural farm crops (A and B), and
also three flushes (I, II, III). During the crop cycle, mushrooms were harvest in a
rhythmic pattern of breaks or flushes that occur at approximately seven days intervals.
After two flushes, production declines rapidly and a grower must decide to terminate
the crop and start a new or face dwindling harvest of mushrooms from each flush.
Table 9. Overview of experimental design used in the procedure, taking into consideration the
sampling stage for each of the different A. biosporus types (cycle stage), according to crop type
and growing tunnel (house).
Sampling stage
Cropping
Flush I Flush II Flush III
A.bisporus cycle stage
BB CC F BB CC F BB CC F
Crop A
House 1 1 1 1 1 - - 1 2 -
House 16 3 1 1 1 1 - 1 1 -
House 17 - 1 2 1 1 - 1 1 -
Crop B
House 1 1 1 1 1 - - 1 2 -
House 16 1 1 1 1 1 - 1 1 -
House 17 - 1 2 1 1 - 1 1 -
n 6 6 8 6 4 0 6 8
n Total 44
5.4. Moisture
The moisture content of the edible mushroom samples was determined, by the
AOAC method, approximately 3 g of each A.bisporus sample were dried in an oven
at 105 ºC overnight (Ouzouni, Veltsistas et al. 2007).
Figure 11. A. bisporus samples from right to left: Baby Buttons (BB); Closed cups (CC) and
Flats (F).
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5.3. Selenium Determination and Sample Preparation
Before freezing the samples at -4ºC whole mushrooms specimens were
mechanically cleaned of soil, rinsed with deionised water and freeze-dried. The
samples defrosted in a petri dish and cutted in a little smithereens weight
approximately 60g of wet sample and left to dry them overnight in the oven at 100ºC.
Three grams of dried A.bisporus were weighed out and added fifty milliliters of HNO3
(69%) where the contents were swirled. The solution were heating up in a hot plate at
120ºC, and after 5 min omitted very strong orange/brown fumes and a large quantity of
gas due to a possible ethanol in our samples coming into contact with nitric acid. A
large quantity of foam was also produced in the initial stage of digestion; this later on
was evaporated in the fumes. The content of dried mushroom samples was reduced to
near dryness. After 40min heating up the development of mushroom digestion, when
was achieved a clear orange solution, were stopped heating. When the liquid
mushroom digestion were cooled ate room temperature other 50 milliliters of nitric acid
were added, and was obtained a final mushroom solution approx 64 ml. Original
mushroom solution was done by adding 15 ml of digest mushrooms in a 100ml
volumetric flask and topped up with ultra-high-purified water. Three replicates of each
sample were prepared. Working diluted solutions 1:10 were prepared in triplicate from
the previous ones and analyze the GFAAS.
Figure 12. Schematic experimental design of sampling. For each A.bisporus were done 3
digestions and in each digestion were done 3 replicates. (e.g.: D1= Digestion nº 1; S1D1R1 =
D1
•S1D1R1
•S1D1R2
•S1D1R3
D2
•S1D2R1
•S1D2R2
•S1D2R3
D3
•S1D3R1
•S1D3R2
•S1D3R3
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Sample nº1, digestion nº1 and Replication nº1). “D” means digestion and “R” replication. In the end
for each mushroom sample were made 9 analyses.
5.5. Standard Preparation and calibration curve
Selenium Atomic Absorption Standard (Aldrich) and Nitric Acid (69% Merk) and
ultra-high-purified water were used. Deionized water was prepared with SGS water
purification system and ultra-higth purified water was obtained using a Milli-Q
Academic System. The distribution of selenium in A.bisporus were carried out in a
Varian Atomic Absorption.
Working standard solutions of selenium at 1000 ppm were prepared by serial dilution of
the commercially available Atomic Absorption Standard at 1002 µg/l of Se wt % nitric
acid. External calibration curve were constructed by plotting the UV emission at
(196nm) versus the amount of selenium using (100; 200; 400 and 600 ppm) as
standards, and the atomic absorption software gave the values of slope, along the
intercept and correlation coefficient for each calibration curve.
5.6. Graphite Furnace Atomic Absorption Spectrometer
conditions
Quantitative GFAAS analyses were performed on a Varian atomic absorption
spectrometer with graphite tube analyzer AA 240 G with the GTA 120 Graphite tube
atomizer and PSD 120 programmable sample dispenser. A Ultra AA Selenium Varian
lamp were used in one of the supporting positions. High purity argon was used as
carrier gas. Equipment was supplied with SpectraAA Base software. Table 9 shows
the conditions applied in Selenium method previously established on Secptra AA
software.
Table 10. Conditions of Se method defined on GFAAS.
GFAAS Conditions
Method Se
Instrument Type Furnace
Calibration Mode Concentration
Conc.Units µg/L
Replicates Standard 2
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Replicates Sample 2
Wavelength 196 nm
Lamp current 10.0 mA
Standard 1 100 µg/L
Standard 2 200 µg/L
Standard 3 400 µg/L
Standard 4 600 µg/L
Resolope Rate 50
Resolope Lower Limit 75%
Resolope Upper Limit 125%
Recalibration Rate 40
Calibration Lower Limit 20%
Calibration Upper Limit 150%
Total Volume 15 µL
Sample Volume 10 µL
Bulk Conc 1000 µg/L
The most advanced and used high sensitive sampling technique for atomic
absorption is graphite furnace. In this technique, a tube of graphite was located in the
sample compartment of the AA spectrometer, with the light path passing through. A
small volume of sample solution was quantitatively placed into the tube, normally
through a sample injection hole located in the center of the tube wall. The tube was
heated through programmed temperature sequence (Table 10) until finally the analyte
present in the sample was dissociated into atoms and atomic absorption occurs.
As atoms were created and diffuse out of the tube, the absorbance rises and falls in
a peak-shaped signal. The peak height or integrated peak area was uses as the
analytical signal for quantification. As described on table 10, the samples were
atomized in a very short period of time, concentrating the available atoms in the heated
cell and resulting in the observed increased sensitivity. The graphite furnace is much
more automated than the other techniques. Even though, this technique uses only
microliter sample volumes, the small sample size is compensated by long atom
residence times in the light path. Heating programs can be very sophisticated, the
FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption
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entire process was automated once the sample had been introduced and the furnace
program initiated.
Table 11. Heating program of the graphite tube atomizer.
The above temperatures were provided in the graphite furnace software (Spectra
AA) without further optimization. The used ramp times correspond to minimum values
to provide the highest heating rate. This increases the residence time of the atomic
vapor in the furnace, maximizing sensitivity and reducing some interference effects. At
the beginning of this step, the spectrometer read function was triggered to start the
measurement of lights absorption.
5.7. Statistical Analysis
The entire data were analyzed by multivariate analysis of descriptive statistics
(minimum, mean, median, maximum, and standard deviation) were calculated for the
concentrations of Selenium. Significant differences in the Se contents between houses,
flush, crop and type of mushroom were evaluated by one-way ANOVA with mixed
effect split-splot design (p< 0.05) where house was a random factor nested to crop. For
graphical displays, boxplots, a graphical analogue of analysis of variance were
performed. All statistical analysis was performed using the SPSS (Version 20) for
Windows.
Step Temp. (ºC) Ramp time (s) Flow (L/min)
1 85 5 3
2 95 40 3
3 120 10 3
4 1000 5 3
5 1000 1 3
6 1000 2 0
7 2600 0.8 0
8 2600 2 0
9 2600 2 3
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The main steps of the entire experimental procedure are represented in figure 13.
Figure 13. Schematic representation of main seven steps from the experimental procedure.
1.A.bisporus fresh sample in individual plastic punnets
2. Defrost and cut samples to dry them in the oven overnigth at 100 ºC.
3. A.bisporus dried sample.
4. Sample digestions a combination between 3g of dried mushroom sample and 100 ml of HNO3 at around 120ºC in a hot plate.
5. Mushroom samples dilutions in 1:10 in ultra purified water.
6. Samples ready to analyze in GFAAS auto sampler.
7. Statistical Analysis - ANOVA with mixed effect split-splot design (p < 0.05)
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6. Results and Discussion
6.1. Method development – Sample digestion
The initial step in analytical methods involving biological matrices as well as
Agaricus bisporus usually involves destruction of the sample and conversion of the
elements to forms suitable for analysis (Campbell 1984). In the case of selenium this is
an important stage in the analysis, because it is a trace element with easy volatilization.
For this reason dry ashing isn’t preferential. That’s why our selenium analysis was
performed with dried Agaricus bisporus samples overnight at 100ºC. Preferred
digestion mixtures involved combinations between approximate 3g of A.bisporus (DW)
and 100ml of nitric acid in a single glass tube at 120ºC during 40 min (until achieved a
clear solution). The volume of nitric acid was quite high, for erase the probability of
charring occurrence during digestion due to the volatility of nitric acid, which would
have a negative effect on selenium recovery. However the high amounts of acid used,
make the method less economic and with a high environmental impact.
Selenium in biological tissues of A.bisporus reacts with nitric acid solution and the
rate of reaction decreases with acidity. Reaction rate was reasonably fast but the
solution remains sufficiently acidic to retain most amount of selenium in solution.
Several practical advantages were found using this digestion experimental procedure:
only one acid (HNO3) was used for digestion and there is no potentially explosive
reaction from perchloric acid with a single tube for digestion, thus minimizing errors and
time spent with manipulation. On the other hand time needed for digestions is long
when compared with other methods previously suggested in the literature (Campbell
1984), for example special fume cupboards or microwaves and Teflon digestion
vessels (Costa-Silva, Marques et al. 2011). According with our lab conditions not a
large number of digestion assays can be routinely performed (in each day of
experience were possible to carried out around 6 digestions from a total of 132
digestions [from each 44 A. bisporus, were being done 3 digestions, that counts 132
digestions in total, plus from each 132 digestion were performed 3 diluted replications,
in total were 396 A.bisporus were analyzed]).
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6.2. Method development – GFAAS calibration
The sensitivity of graphite furnace atomic absorption makes it is the obvious choice
for trace metal analysis applications (Beaty 1978). Routine determinations at µg/L level
for selenium make it ideal for quantification of this trace element (Se) in mushrooms.
The microliter (µL) sample sizes used offer additional benefits where the amount of
sample available for analysis is limited. Calibration is a common and essential step in
analytical methods, it is essential that analysts have a good understanding of how to
step up calibration experiments and how to evaluate the results obtained.
To formulate an accurate calibration equation, it is very important to select a
wavelength at which absorption assigned to the target of selenium can be observed.
The selection of a wavelength for formulating a calibration equation of selenium content
was investigated (EPA methods 7741A). Absorption assigned to selenium was
observed at 196 nm on the raw spectra.
Under the ideal experimental conditions (automatic defined by GFAAS Se method,
described in Spectra AA software), calibration curve for determination of Se were
constructed (Figure 14).
Figure 14. Calibration curve for Se standards. Absorbance of the analyte versus Se concentration
at (100, 200, 400 and 600µg/L).
y = 0.0003x + 0.0184 R² = 0.9977
0
0,05
0,1
0,15
0,2
0,25
0 200 400 600 800
Abs
Se µg/L
Se Calibration curve
Se Calibration curve
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Figure 14 clearly represent a linear regression of the integrated absorbance
signal for selenium concentrations in the range of 0.0231 – 0.2049 absorbance. The
method was investigated in triplicate and was found to be linear in the range 100 – 600
µ/L. The calibration standards were evenly spaced over the concentration likely to be
encountered for mushrooms samples, and the calibration standards run in triplicate.
Between every 40 samples being analyzed in GFAAS, the equipment made a
recalibration by itself, and all the recalibrations followed a linear calibration curve.
Precision in the above concentration range was around 3.4% RSD which is analytically
acceptable.
6.3. Moisture content
Knowledge of the water distribution in the mushroom white buttons or A.bisporus is
of major interest for studying the postharvest senescence of this economically
important crop (Donker, Van As et al. 1997).
Watering or irrigation is one of the most delicate operations in mushroom growing.
The increase in the weight of the mushroom from pinning to maturing is related to the
rapid uptake of water from the casing and the compost. As the mushroom matures
during a flush, its weight gain is attributed to accumulation of nutrients and water from
substrate (Beyer and Extension 1997).
The amount of water in mushrooms was expressed in percentage by weight of
water in mass, i.e. as a percentage of total dry weight of mushrooms samples (Table
12).
Table 12. Moisture content of three different types of A.bisporus.
Type of A.biporus % Moisture
Baby Buttons 90.0 ± 1.59
Closed Cups 91.0 ± 1.31
Flats 91.09 ± 0.90
All the samples in the present study maintained moisture content values around
90% (Table 12). This was in agreement with findings reported for mushrooms by
(Ouzouni, Veltsistas et al. 2007) . In fact, mushrooms are one of the highest water-
containing foods. There is no significantly a difference in moisture content between
types of mushrooms in the same specimen A.bisporus. All of them baby buttons,
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closed cups and flats are around 90% water quantity, and there is no differences in
water contents between mushrooms within flush number, crop type our house number.
6.4. Selenium content of Irish Agaricus bisporus
The cultivated mushroom A.bisporus our usually named as white button
mushrooms, is the most consumed mushroom in Ireland, as well as worldwide.
Commercially, these studied specie can be found fresh, frozen, either whole or sliced
(in the case of closed cups), flats and baby buttons are always available in whole way
in Irish market, due to them size. The 44 commercial and edible mushrooms in present
study were fresh and whole mushrooms were considered in this research.
All the samples in this study were organized according with the effect of growing
conditions on them. These growing conditions refer to a cropping stage, flush level, and
tunnel (house) where mushrooms grown. There are two cropping stages (A and B),
and three flushes levels (I,II and III), in all types of A.bisporus “BB”; “CC” and “F”, with
the exception of the flats, in which this type are represented only in flush I. Because of
this fact data analysis was conducted in order to identify possible differences in
selenium concentrations between crop A and B, in baby buttons and closed cups
during 3 flushes, and no comparisons within flats (Table 13).We decided to separate
the flush effect and it was analyzed only in two types of mushrooms in “BB” and “CC”,
(Table 13); and in the other perspective analyze the effect of selenium in 3 types of
mushrooms “BB”, “CC” and “F” (Table 14). The total contents of Se in A.bisporus
analyzed are shown in table 13 and table 14. Mean, standard deviation and number of
samples are represented. The results are expressed in micrograms of Se per gram (dry
weight) for all samples. The concentrations found in the present work are compared
with those reported previously in the literature.
The selenium levels (µgSe/g dry mushrooms) found in both of types (“BB” and
“CC”), is considerably higher along the flush level, i.e., the amount of selenium
increase during the flush levels, selenium contents in flush III was considerably higher
than selenium contents in flush I, independent of the type of mushroom. The selenium
levels in A.bisporus are mighty affected by flush.
Mushrooms appear in flushes i.e. a flush of mushroom will appear and be picked in
3-5 days, and then a gap of 5 days will lapse before the next flush appears. Growers
generally take 3-5 flushes from each crop, before the crop finishes and the house is
emptied (Beyer and Extension 1997).
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Table 13. Mean value (µgSe/g Dry mushroom) and standards deviation of Selenium content in Irish Agaricus bisporus as a function of crop type, growing house
and flushing stage, for baby buttons and closed cup mushrooms.
A. bisporus cycle stage Crop A Crop B
Type Flush House 1 House 16 House 17 House 1 House 16 House 17
Baby Button
Flush I 7.5 ± 3.0 (9) 52.1 ± 22.8 (25) nd 9.4 ± 1.9 (9) 56.2 ± 10.1 (8) nd
Flush II 62.1 ± 17.1 (9) 58.2 ± 21.9 (9) 10.0 ± 0.9 (6) 63.8 ± 30.8 (9) 69.1 ± 16.5 (9) 14.1 ± 2.7 (7)
Flush III 65.0 ± 33.4 (6) nd 64.8 ± 33.7 (9) 53.2 ± 28.6 (6) 54.1 ± 24.2 (6) 67.1 ± 25.6 (8)
Closed Cup
Flush I 39.5 ± 1.5 (8) 2.8 ± 0.6 (9) 54.5 ± 19.5 (9) 42.4 ± 9.1 (8) 14.7 ± 11.7 (9) 62.2 ± 29.4 (8)
Flush II nd 62.5 ± 17.7 (9) 2.6 ± 0.3 (8) nd 59.7 ± 24.0 (9) 14.5 ± 6.2 (9)
Flush III 57.5 ± 28.2 (9) 61.0 ± 19.8 (6) 68.8 ± 31.6 (9) 54.9 ± 28.5 (15) 51.5 ± 20.4 (6) 62.4 ± 26.2 (9) nd – no data
Table 14. Mean value (µgSe/g Dry mushroom) and standards deviation of Selenium content in Irish Agaricus bisporus as a function of crop type and growing
house, for baby buttons closed cup and flat mushrooms from the first fructification cycle: flush I.
nd – no data
A.bisporus cycle stage Crop A Crop B
Type House 1 House 16 House 17 House 1 House 16 House 17
Baby Button 7.5 ± 3.0 (9) 52.1 ± 22.8 (25) nd 9.4 ± 1.9 (9) 56.2 ± 10.1 (8) nd
Closed Cup 39.5 ± 1.5 (8) 2.8 ± 0.6 (9) 54.5 ± 19.5 (9) 42.4 ± 9.1 (8) 14.7 ± 11.7 (9) 62.2 ± 29.4 (8)
Flat 27.1 ± 7.9 (6) 26.1 ± 3.9 (8) 50.4 ± 26.1 (17) 23.0 ± 7.8 (9) 32.4 ± 3.6 (9) 13.4 ± 5.2 (9)
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This fact can be a possible reason for the higher selenium concentrations during
the flush, because this trace element (Se) is bioavailable in the compost, it means that
how much longer mushrooms keep in contact with crop, it will expected that more
selenium amount will be absorbed by the fungi. Consequently, mushrooms from flush
III are longer in contact with compost when compared with mushrooms from flush I and
II.
Comparing the values found for the amount of selenium in three different types of
mushrooms (Table 14), it can be observed that is a trend for a higher selenium
concentrations (µgSe/g dry mushrooms) in Baby buttons (the smaller type), although
this effect it’s not quite clearly (Table 14), e en though this fact will be evident
demonstrate bellow in ANOVA.
From table 14, in edible Irish mushrooms, the highest Se level was found as 69,1
2.2 ± 16.5 (µgSe/g dry mushrooms) for baby button type, collected in flush II and in
house 16 , followed by closed cup collected in house 17 in flush III in which selenium
amount was 68.8 ± 3.6 (µgSe/g dry mushrooms). The lowest Se concentration was 2,8
± 0,6 in closed cup from house 17, and flush I. The large standard deviation present in
majority of selenium concentrations, indicates that the data values are far from mean,
and data points are spread out over a large range of values. One possibility to improve
this dispersion could be increasingly the number of samples in each factor of analysis.
The trace element (Se) contents in cultivated mushrooms of the specie A.bisporus
depend on the ability of the specie to extract selenium from the substrate, and on the
selective uptake and deposition of selenium in tissues (Ayhan 2001). An interesting
aspect of our study is that different types of mushroom sample from the same specie
differ considerably in their selenium content, and the highest selenium concentrations
were found in flush III.
The selenium content of the Irish fresh A.bisporus are much higher than selenium
content reported in other different locations as well as Portuguese fresh A.bisporus in
a range of 0.637 – 1.249 mg/Kg DW (Costa-Silva, Marques et al. 2011) or Italian fresh
A.bisporus which were reported a selenium amount of 3.40 mg/kg fresh weight
(Cocchi, Vescovi et al. 2006). Unfortunately, data on the Se content in mushrooms
consumed in Ireland have not been reported at this time, so it is not possible to carry
out a comparison of the results obtained in this study.
The relative portion of selenium in the environment varies according with the
geographic location, with native substrate, climatic conditions and vegetation cover. In
central US, for example, there are regions in which plants contain Se levels 10 times
higher than toxic level, while Se levels in plants in eastern and western US are low
(Kubota, Allaway et al. 1967). In Ireland, toxic levels was registered in some counties
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according with (Rogers, Arora et al. 1990), as like Carlow, Dublin, and Limerick, but
there not yet data reporting Se contents in Mongham countie.
.
6.5. Effect of production and cycle factors on selenium content
The present study was designed to evaluate two main effects of Selenium contents
in a natural and fresh product such as A.bisporus. The first goal was to investigate the
effects on growth performance in mushrooms, as well as flush effect considering house
a random factor. Up to this point, we have treated all categorical explanatory variables
as if they were the same importance. There was considered two fundamentally
different sorts of categorical explanatory variables: the fixed effects (i.e. flush, crop and
type of mushrooms) and random effects (number of house or tunnel where the samples
growth). ANOVA analysis with a split- plot mixed effect was performed. In simple terms,
a split-plot experiment is a blocked experiment, where the blocks themselves serve as
experimental units for a subset of all the four factors in A.bisporus analysis. On
Selenium concentration present in A.bisporus it is a marked heterocedasticity thus log
|Se| have been used to stabilize variance across the groups. The distinction is best
seen in tables below (Table 15, 16, 17).
We choose to express the ANOVA data analysis in two different units of Selenium
concentration, (µg/g Se Fresh Mushroom) and (µg/g Se Dry Mushroom).
6.5.1. Selenium content in fresh A.bisporus expressed in µg/g fresh
mushrooms
The analysis of split-plot experiment is more complex than that for a completely
randomized experiment due to the presence of both split-plot and whole plot random
errors. In statistical data analysis, the set of 4 variables (flush, crop, type and house)
had sub-groups (Flush I,II,II; crop A and B; Type BB,CC and F; House 1,16 and 17,
respectively ) which have different variability from others, and because of this fact
selenium concentration in commercial edible mushrooms is heterocedastic. The
variability was quantified by the variance. The existence of heterocedasticity in
selenium concentration of A.bisporus invalidated statistical tests of significance that
assume that the modeling errors are uncorrelated and normally distributed.
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The cultivated fresh A.bisporus (white mushroom) is the most consumed in
Ireland, and also in worldwide. The selenium contents of commercial A.bisporus are
presented in the following tables, where 95% confident interval was considered.
Table 15. Significance values for the mixed-effect split-plot ANOVA with two factors for log10 |Se| fresh mushroom.
Source /Factor p value
Crop (main block) 0.593
House (random factor nested under crop) 0.038
Type of mushroom 0.007
Flush 0.000
Type of Mushroom * Flush 0.036
Bold values are significantly at p ≤ 0.05
In the above table was a clearly marked effect of the house, type of mushroom
and flush across the Selenium concentration in Irish A.bisporus. It means that the
blocked factor “crop” which is di ided in two categories (Crop A, and B), demonstrate to
be a non decisive factor for the amount of selenium and have mixed effect with house,
type of mushroom and flush. Similarity to the previous studies conducted with
descriptive statistics about Selenium concentrations (µg/g Se DW), an examination of
table 16 also demonstrate that flush effect is extremely marked, and it is clear shown
specifically in two types of A.bisporus – baby buttons (BB) and closed cups (CC).
Baby buttons and closed cups, are available in three flushes, otherwise flats are
only cultivated in the first flush. For this reason was performed a single-step multiple
comparison to find which means are significantly different from one another between
baby buttons and closed cups during the flush number. A tuckey’s test were performed
in order to identify honestly significan difference.
Table 16. Selenium content (µg/g Se Fresh Mushroom) in baby buttons and closed cups, expressed values during the flush number.
Flush number Mean Type of Sample Mean
Flush I 2.5b Baby Button 2.3
Closed Cups 2.3
Flush II 3.0b Baby Button 3.7
Closed Cups 2.0
Flush III 5.5a Baby Button 6.2
Closed Cups 5.4
a,b – homogeneous groups according to the multiple comparison Tuckey test at 95% confidence
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Values are significantly at p ≤ 0.05. Means for groups in homogenous subsets
was displayed, based on observed means. The error term is mean square (Error) =
0,062, with α =0.05.
The highest selenium concentration in fresh mushroom was 6.2 µg/g Se Fresh
Mushroom in baby buttons collected in flush III. Selenium content in Baby buttons
ranged from 2.3 - 6.2 µg/g Se Fresh Mushroom corresponding to Flush I and III
respectively, and closed cups selenium concentrations varies in a range of 2.3 – 5.4
µgSe/g Fresh Mushroom also corresponding to the first and last flush respectively.
That is an evident higher selenium content in the last flush (flush III) in both of type of
mushromm (Baby button and closed cup).
Another way to illustrate the flush effect in selenium concentration µg/g Se
Fresh Mushroom is resorting to an interaction plot (Figure 15).
Figure 15. Estimated marginal means for log10 |Se|, in fresh mushrooms, depicting the interaction effect between flushe order and type of mushrooms.
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In this design with two factors, the marginal means for one factor are the means for
that factor averaged across all levels of the other factor
The above model represented in figure 15, shown a design where Flush III have a
tendency to present a higher selenium concentration (µg/g Se fresh mushroom), In
flush II there is an evident distance between Baby button and closed cup, which define
the interaction effect between type of mushroom and flush. Through the interaction
graph, we can observe that baby button get a trend to exponentially increase selenium
concentrations during the flushing evolution.
An alternative method to illustrate the flush effect on selenium concentration is the
box plot display. Box plot shows more than just four split groups. In figure 16 we also
observed which way the data sways. Is an evident fact that there is more selenium
concentration in A.bisporus from flush III, than selenium concentration in A.bisporus
which was collected in flush I. The above box plot gave us a good overview of the
data’s distribution.
Figure 16.Box plot displays of the selenium content (µg Se/g Fresh Mushroom) distribution within
Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house
number .
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6.5.2. Selenium content in A.bisporus in dry mushrooms
Another gourmet trendy is the consumption of white mushrooms sliced dried,
and it also commercially available in the common markets.
In this experiment, treatments were applied only to groups of experimental
observations rather than separately to each observation. In this case were two nested
groupings of the observations on the basis of treatment application, and this known as
a split- design. The sampling fraction was the number of values in the sample divided
by the number of values all A.bisporus samples. In ANOVA the notion of random
slopes is functionally equivalent to the notion of a treatment – by – subject interaction.
Most a Selenium concentration expressed in µgSe/g Dry Mushroom (DW) was
performed.
On table 18, are represented the p values obtained from split-plot analysis of
log10 |Se|, in the four factors (Crop, number of house,type of mushroom, flushing and
type of muhroom within flush).
Table 17. Significance values for the mixed-effect split-plot ANOVA with two factors for the log10
|Se| dry mushroom
Source /Factor p value
Crop (main block) 0.677
House (random factor nested under crop) 0.018
Type of mushroom 0.027
Flush 0.000
Type of Mushroom * Flush 0.056
Bold p-values are significant at p ≤ 0.05
The above results demonstrated significant differences in a confidence interval
of 95% for three factors in studied of selenium amounts in Irish A.bisporus. Significantly
sources was house, type of mushroom and flush. In other words house number, type
of A.bisporus and flush number were sources which have an influence on the selenium
contents.
As well were shown on (6.5.1) for fresh weight of A.bisporus, the same analysis
were performed for A.bisporus dry weight. The selenium content (µgse /g mushroom
DW) (Table 19). The selenium content (µgse /g mushroom DW) in irish edible
A.bisporus shown an homogeneous attitude during the flushing. A.bisporus which was
collected in flush III, were more rich in selenium contents than A.bisporus collected in
flush I and II (Table 19)
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Table 18. Selenium content (µgSe/g Mushroom DW) in baby buttons, expressed values during the flush.
Flush number Mean Type of Sample Mean
Flush I 24,0b Baby Button 22.6
Closed Cups 21.8
Flush II 29,7b Baby Button 37.8
Closed Cups 19.6
Flush III 52,7a Baby Button 58.0
Closed Cups 53.8
a,b – homogeneous groups according to the multiple comparison Tuckey test at 95% confidence
This is an interesting fact in the sense of mushrooms from flush III are
considered the fresh products with the lowest quality, while mushrooms from flush I are
considered the best fresh products for consumer.
The estimated marginal means shown the mean response for each factor
adjusted for any other variables in the model (figure 17).
Figure 17. Estimated marginal means for log10 |Se|, in dry mushrooms, depicting the interaction effect between flushe order and type of mushrooms.
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The above general linear model is a flexible statistical model that incorporates
normally distributed dependent variables and categorical or continuous independent
variables. This procedure is very useful , where by a design, allows a detail discussion
the two types of sums of suquares, estimated marginal means.
On the figure 18, is presented an overview, where is shown a great and simple
illustration of selenium content in commercial Irish mushrooms (µg Se/g mushroom
DW) within and between the four factors in study.
Figure 18. Box plot displays of the selenium content (µg Se/g dry Mushroom) distribution within
Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house
number.
6.5.3. Effect of growing stage on selenium content of fresh
mushrooms
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Based on data from flush I, inspection of the effect of growing stage: BB,CC, and F
on the selenium content of fresh mushrooms was performed based on a mixed-effect
split-plot ANOVA
The accumulation of selenium in A. bisporus could be groups/factors – specific and
thus assume that house was a random factor nested under crop, and was significantly
different at 95% interval of confidence.
Table 19. Significance values for the mixed-effect split-plot ANOVA with one factor for log10 |Se| fresh mushroom.
Source /Factor p value
Crop (main block) 0.731
House(random factor nested under crop) 0.000
Type of mushroom 0.052
Bold values are significantly at p ≤ 0.05
Type of muhroom – dependent selenium concentrations in the fruiting bodies of
A.bisporus were observed (table 22). Table 22 shows that the amount of selenium
accumulated in the samples studied varies according with the type, i.e. selenium
amounts of baby buttons, closed cups and flats are quite different among them.
Flats were the mushroom type which accumulate a lowest selenium amount. On
the other hand baby buttons was the A.bisporus type which allowed more selenium
accumulation. Baby buttons are in size, the smallest type of mushrooms compare with
closed cups and flats. The falts or also called Portobello are the biggest ones in size.
Unfortunately there is no available data which we can compare our data. There are
data values which compare selenium amounts between species, but between types of
mushroom within the same sample there aren’t.
Table 20. Selenium concentration (µg/g Se Fresh Mushroom) in commercial A.bisporus 3 types of mushroom in study.
Type of Sample Mean Std Error
Baby Button 31.6 0.037
Closed Cup 21.7 0.032
Flat 23.4 0.031
Values are significant at p ≤ 0.05
Figure 19. Interaction effect between one factor - type of mushrooms (BB, CC and F).
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The present ANOVA model is based on the assumption that there is a single
error term. But in the case of our study nested experiments like split-plot design were
performed, where data was gathered at different spatial scales, there is a different error
variance for each different plot size.
The variance was likely different at each level of this nested analysis possible due to:
The readings in GFAAS may have differed because of variation in selenium
concentrations in each A.bisporus sample;
The pieces of mushrooms used in digestion procedure weren’t homogeneous.
6.6. Irish A.bisporus contribution to the Se daily intake
The food and nutrition database is quite important for an accurate evaluation of
nutrient intake from dietary intake surveys. Plant species and fungi species do not
require Se for growth and can be very low in Se, in contrast to animal species, for
which Se is an essential nutrient, and which will not survive if tissue levels are too low
(Murphy and Cashman 2001). Most animal foods such as fish shellfish, meats, and
eggs have a high selenium content, which has previously been reported in several
research studies (Barclay, MacPherson et al. 1995; Murphy and Cashman 2001;
Sirichakwal, Puwastien et al. 2005; Navarro-Alarcon and Cabrera-Vique 2008). On the
other hand, vegetables and fruits in general are assumed to contain low levels of
selenium unlike foods of animal origin.
Comparing the values found for the amount of selenium in different types of
A.bisporus with the dietary reference intake (DRI) of selenium for healthy adults, man
and woman (55µg of selenium represented the dietary allowance (Directorate 2000)), it
can be observed that commercial Irish A.bisporus can be considered as a good
selenium source in the Irish diet. The average of quantity of mushrooms per person per
day was estimate in 9.72 g in 1999 according with Pan – European Food data bank
based on houselhold budget surveys. Fresh A.bisporus are considered one of the main
foods in Irish meals, they can be included even in the traditional Irish breakfast.
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7. Conclusions
Selenium is an element which plays an important role in human nutrition and
metabolism
A sensitive, reproducible and relatively simple GFAAS method was developed to
screen and quantify selenium in A.bisporus, the applicability of the method was
demonstrated by analysis of several different types of A.bisporus samples.
The conditions of GFAAS chosen guaranteed a good resolution and identification of
the essential trace element – Se. A suitable sample preparation including acid
digestion was successfully optimized allowing a further application of the analytical
method.
Our data analysis were divided in two sets, where the aim was shown the flush
effect on selenium concentrations, and in the other hand a comparison between types
of A.bisporus only in flush I. Data proved that flush have a hardly effect on the selenium
concentrations (µgse/g mushrooms DW), and in relation to type the of mushrooms
baby buttons shown a trend to accumulate more Selenium contents.
Finally, all the general and specific objectives were successfully accomplished.
The work present in this master thesis could be extended and improved taking
some considerations:
More number of mushroom samples should be use in order to reduce the
standard deviation between analysis;
Increase the factors in study, i.e., add more houses and cropping system to
the sampling experimental design;
Would be interesting a study focused on the selenium contents in
soil/compost where mushrooms growing up, for the reason that selenium
contents in fungi depend of selenium bioavailability in the compost.
In order to extend the study more heavy metals could be analyzed as well
as Arsenic; Copper; Iron; Fe; cadmium; mercury and lead, keep using the
same analytical method GFAAS which have a adequate sensitivity for heavy
metals.
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Annexes
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Figure 20. Box plot displays of the selenium content (µg Se/g dry Mushroom) distribution within
Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house number.
Figure 21. Box plot displays of the selenium content (µg Se/g fresh Mushroom) distribution within Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house
number.
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Figure 22. Box plot displays of the selenium content (µg Se/g dry Mushroom) distribution within
Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house number.
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Figure 23. Box plot displays of the selenium content (µg Se/g dry Mushroom) distribution within
Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house number.
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Figure 24. Box plot displays of the selenium content (µg Se/g dry Mushroom) distribution within Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house
number.
Figure 25. Box plot displays of the selenium content (µg Se/g dry Mushroom) distribution within
Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house number.
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Figure 26. Box plot displays of the selenium content (µg Se/g fresh Mushroom) distribution within Irish A.bisporus mushroom according to flush order, type of mushroom, type of crop and house
number.