Determination of selenium content in Irish commercial ... · Determination of selenium content in...

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

http://johnmaxwellonleadership.com/

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

iv

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

v

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.

FCUP Selenium speciation in Irish commercial Agaricus bisporus by Atomic Absorption

vi

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)


vii

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


viii

NRI - Reference Nutrient Intake

NSPs – Polysaccharides

SeCys - selenocysteine

SeP - Selenoprotein

UK – United Kingdom

US – United States

RSD – Relative Standard deviation

WHO – World Health organization


ix

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


x

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


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 21. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G FRESH
















xi

FIGURE 26. BOX PLOT DISPLAYS OF THE SELENIUM CONTENT (µG SE/G FRESH




xiii

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


xiv

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


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


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


xv

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


xvi

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

FCUP Determination of selenium content in Irish commercial Agaricus bisporus by Atomic Absorption

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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)


20

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.


21

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)).


22

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).


23

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)


24

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


25

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.


26

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


27

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)


28

(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.


29

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).


30

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


31

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


32

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)


33

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


34

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)


35

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,


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)


37

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).


38

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;


39

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).


40

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.


42

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


43

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


44

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)


45

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).


46

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,


47

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)


48

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).


50

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


52

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


53

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).


54

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


55

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


56

Replicates Sample 2

Wavelength 196 nm

Lamp current 10.0 mA

Standard 1 100 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


57

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


58

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)


60

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]).


61

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


62

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,


63

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).


64

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)


65

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


66

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.


67

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


68

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.


69

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 .


70

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



House (random factor nested under crop) 0.018


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)


71

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.


72

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


73

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.



House(random factor nested under crop) 0.000


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).


74

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.


75

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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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 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.




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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.

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