MITIGATION OF AFLATOXIN B1 BY

LACCASES FROM TRAMETES VERSICOLOR

Laure DEGROOTE Student number: 01305659

Promoter: Prof. Dr. Apr. Sarah De Saeger

Co-promoter: Prof. Dr. Chiara Dall’asta

Department of Bioanalysis Ghent

Commissioners: Dr. Marthe De Boevre and Dr. Arnau Vidal Corominas

Academic year: 2016 - 2017

MITIGATION OF AFLATOXIN B1 BY

LACCASES FROM TRAMETES VERSICOLOR

Laure DEGROOTE Student number: 01305659

Promoter: Prof. Dr. Apr. Sarah De Saeger

Co-promoter: Prof. Dr. Chiara Dall’asta

Department of Bioanalysis Ghent

Commissioners: Dr. Marthe De Boevre and Dr. Arnau Vidal Corominas

Academic year: 2016 - 2017

COPYRIGHT

“The author and the promoters give the authorization to consult and to copy parts of this

thesis for personal use only. Any other use is limited by the laws of copyright, especially

concerning the obligation to refer to the source whenever results from this thesis are cited.”

June 1, 2017

Promoter Author

Prof. dr. Apr. S. De Saeger Laure Degroote

SUMMARY

The presence of mycotoxins in food and feed is a well-known problem for human

health and economy. This paper aimed at investigating the mitigation of aflatoxin B1 by a

multicopper oxidase namely, laccase from Trametes versicolor. More specifically, the

potential of laccase in the decontamination of aflatoxin B1 was studied in presence of a

chemical mediator (ABTS), a natural polyphenol (rutin hydrate), a blank maize extract or in

absence of additional compounds. The remaining level of aflatoxin B1 was determined by

the UHPLC-MS/MS or HPLC with fluorescence detection for approximately 48 hours. The

experiments were performed in vitro at 35 °C in citrate buffer solution. Moreover, efforts

were made to elucidate the reaction product(s).

A statistically significant reduction in the AFB1 level due to the laccase enzyme, has

only been observed if a mediating system was present. Removal of 97.37% and 93.39%

was achieved in 46 hours for the samples compared to the controls (without laccase) when

using ABTS or ABTS and rutin hydrate together as mediating systems. For the samples

with maize extract, a decrease of 51.44 % was observed in 48 hours which may suggest

that a natural mediator or even a combination of mediators may be present in the maize

extract which can trigger the enzymatic reaction. Unfortunately no structure(s) of reaction

product(s) were elucidated during this research.

Further investigation in this field needs to be done in future. It would be interesting

to test the influence of laccases on food or feed products naturally contaminated with

aflatoxin B1. Moreover, the elucidation of eventual reaction product(s), could give relevant

information about the laccase enzyme mechanism, which has to be known before applying

in food and feed. Also toxicity and stability of the reaction product(s) must be determined.

SAMENVATTING

De aanwezigheid van mycotoxinen in voedsel, bestemd voor zowel menselijke als

dierlijke consumptie, vormt een grote bedreiging voor humane gezondheid en economie.

Het doel van deze paper was om de mitigatie van aflatoxine B1 door een oxidoreductase

enzyme, namelijk laccase afkomstig van Trametes versicolor, te onderzoeken. Meer

specifiek werd bestudeerd in welke mate laccase in staat was om het gehalte aan

aflatoxine B1 te doen dalen na toevoeging van een chemische mediator (ABTS), een

natuurlijke polyphenol (rutin hydrate), een blanco mais extract of zonder de toevoeging van

een extra molecule. Alle verschillende reacties werden in vitro uitgevoerd in een

citraatbuffer bij 35 °C. Het gehalte aan aflatoxine B1 werd gemonitord via de UHPLC-

MS/MS of HPLC-FLD gedurende 48 uur. Bijkomend werd ook getracht om eventuele

reactieproduct(en) te identificeren.

Enkel in aanwezigheid van een mediator (e.g. ABTS) voor het enzyme, werd een

statistisch significante daling in aflatoxine B1 waargenomen door laccase. Een daling van

97.37% en 93.39% voor respectievelijk het toevoegen van ABTS en ABTS samen met

rutine werd vastgesteld in een tijdsperiode van 46 uur. Voor de stalen op basis van een

blanco mais extract, werd een statistisch significante daling van 51.44 % in aflatoxine B1

gedetecteerd in 48 uur, wat doet vermoeden dat eventuele mediator(en) of andere

substanties in het mais extract aanwezig waren, die de enzymatische reactie kunnen

triggeren. Helaas werden geen structuren van reactieproducten opgehelderd.

Algemeen kan geconcludeerd worden dat meer onderzoek moet verricht worden

naar het reactiemechanisme van laccase voor aflatoxine B1 vooraleer dergelijke mitigatie

techniek kan gebruikt worden op voeding voor dierlijke en humane consumptie. Hiervoor

is de opheldering van eventuele reactieproducten noodzakelijk. Ook de evaluatie van de

toxiciteit en stabiliteit van reactieproduct(en) dient te gebeuren.

ACKNOWLEDGEMENTS

A lot of people contributed to the realisation of my thesis and in this section I would like to

thank all of them.

In first place, I want to warmly thank Prof. dr. Apr. De Saeger Sarah who made it possible

for me to perform my master thesis abroad.

I also want to show my gratitude towards Prof. dr. Dall’asta Chiara. She made me feel a

part of her research team and was always there to answer questions and give

suggestions.

Moreover I would like to thank postdoc Luca Dellafiora who was giving me relevant

information and advice throughout different experiments. He taught me to be open-eyed

during research without forgetting the things around you.

And last but not least, I am very thankful for being surrounded with such a nice

colleagues and peer students who were very friendly, encouraging, helpful from the first

moment I arrived in Parma! Furthermore, I would like to say ‘thank you’ to my parents,

family and friends for giving me lots of support during the entire 4 months.

TABLE OF CONTENTS

1. INTRODUCTION ......................................................................................................... 1

1.1. MYCOTOXINS...................................................................................................... 1

1.1.1. General introduction .................................................................................... 1

1.1.2. Mitigation of mycotoxins ............................................................................. 2

1.2. AFLATOXINS ....................................................................................................... 3

1.2.1. Aflatoxin B1 .................................................................................................. 3

1.3. LACCASES .......................................................................................................... 5

1.3.1. Characteristics of laccase ........................................................................... 5

1.3.2. Trametes versicolor ..................................................................................... 7

1.3.3. Biological applications ................................................................................ 7

1.3.4. Mediators of laccase .................................................................................... 8

1.3.5. Preliminary research .................................................................................. 10

2. OBJECTIVES ........................................................................................................... 13

3. MATERIAL AND METHODS .................................................................................... 14

3.1. EXPERIMENTAL DESIGN ................................................................................. 14

3.2. MAKING OF CITRATE BUFFER ........................................................................ 15

3.2.1. Chemical agents ......................................................................................... 15

3.2.2. Stock solutions of citric acid and sodium tri-acetate – preparation ...... 15

3.2.3. Combination of stock solutions ................................................................ 16

3.3. SAMPLE PREPARATION .................................................................................. 16

3.3.1. Chemical agents ......................................................................................... 16

3.3.2. Practical implementation ........................................................................... 17

3.3.2.1. Overview sample list .............................................................................. 17

3.3.2.2. Aflatoxin B1 samples and controls ......................................................... 18

3.3.2.3. Aflatoxin B1 samples with ABTS and controls ....................................... 19

3.3.2.4. Aflatoxin B1 samples with rutin hydrate and controls ............................ 20

3.3.2.5. Aflatoxin B1 samples with ABTS, rutine hydrate and controls ............... 20

3.3.2.6. Aflatoxin B1 samples and controls with maize extract ........................... 21

3.3.2.7. Blank samples ....................................................................................... 22

3.4. INTERRUPTION OF REACTION - PROCEDURE ............................................. 23

3.4.1. Chemical agents ......................................................................................... 23

3.4.2. Practical implementation ........................................................................... 23

3.5. ANALYSIS OF SAMPLES .................................................................................. 25

3.5.1. Chemical agents ......................................................................................... 25

3.5.2. UHPLC-MS/MS and HPLC with fluorescence detection .......................... 25

3.5.2.1. (U)HPLC ................................................................................................ 26

3.5.2.2. Tandem Mass Spectrometry (MS/MS)................................................... 27

3.5.2.3. Fluorescence detection (FLD) ............................................................... 29

3.5.3. Statistical analysis ..................................................................................... 30

4. RESULTS ................................................................................................................. 31

4.1. VARIOUS EXPERIMENTS WITH AFLATOXIN B1............................................. 32

4.1.1. Aflatoxin B1 ................................................................................................ 32

4.1.2. Aflatoxin B1 in presence of ABTS ............................................................ 33

4.1.3. Aflatoxin B1 in presence of rutin hydrate ................................................ 34

4.1.4. Aflatoxin B1 in presence of ABTS and rutin hydrate .............................. 35

4.1.5. Aflatoxin B1 in presence of an natural blank maize extract ................... 37

4.2. TENTATIVE IDENTIFICATION OF POSSIBLE REACTION PRODUCT(S) ....... 38

5. DISCUSSION ............................................................................................................ 40

5.1. EVALUATION AFLATOXIN B1 DECONTAMINATION POTENTIAL OF LACCASES ................................................................................................................... 40

5.2. TENTATIVE IDENTIFICATION OF POSSIBLE REACTION PRODUCT(S) ....... 42

5.3. POSSIBLE REAL-WORLD APPLICATION IN FOOD AND FEED...................... 43

6. CONCLUSION .......................................................................................................... 45

7. REFERENCES .......................................................................................................... 47

LIST WITH ABBREVIATIONS

ABTS: 2,2’-azino-di-(3-ethylbenzotiazoline)-6-sulfonic acid

AFBO: Aflatoxin B1-8,9-epoxide

AFB1: Aflatoxin B1

AFM1: Aflatoxin M1

ANOVA: Analysis of variance

ESI: Electrospray ionization

FAO: Food and Agriculture Organization of the United Nations

FLAP: Fingerprint for Ligand And Protein

FLD: Fluorescence detector

GAP: Good Agriculture Practices

HINT: Hydropathic INTeractions

IARC: International Agency for Research on Cancer

MS/MS: Tandem Mass Spectrometry

m/z: mass over load ratio

PPM: Parts Per Million (mg/kg)

Q-TOF-Mass Spectrometer: Quadrupole time-of-flight mass spectrometer

RIC: Reconstructed Ion Chromatogram

TIC: Total Ion Chromatogram

(U)HPLC: (Ultra) High Performance Liquid Chromatography

1

1. INTRODUCTION

1.1. MYCOTOXINS

1.1.1. General introduction

Some fungal species that contaminate crops and commodities are able to produce

low-molecular metabolites in appropriate circumstances. These secondary metabolites,

which may enter food and feed production, are called mycotoxins. The most relevant fungal

species, having this biological activity, are Aspergillus, Penicillum and Fusarium genera.

There are various kind of mycotoxins known with highly diverse organic structures. This is

responsible for the differences in toxicity between several toxins. If consumed for high

enough quantities or for quite a long period of time, mycotoxins can cause cancer, acute

toxicities, developmental disorders etc.. [1, 2]

Because of the major concerns for public health and welfare, many countries have

adopted regulations hereby trying to lower potential dietary intake (Europe: EC No

1881/2006, EU No 165/2010, EU No 105/2010). Beside regulations, there are also

recommendations, which are formed by the European Commision. Harmonizing the

allowed levels of contamination among countries, may avoid worldwide trade frictions. This

harmonization needs to be improved in future. [1, 3]

Beside the caused toxicity, mycotoxin contaminations may also lead to economic

losses. First of all due to the trade frictions, as previously mentioned. Developing countries

might be not competitive compared to industrialized countries because of the lack of

regulatory actions and monitoring plans. This makes it almost impossible for regions like

Africa to meet the imposed maximum contamination levels of industrialized countries. [1]

The presence of mycotoxins may also lead to an increasing cost for toxicosis treatments,

an increasing cost for finding substitute food, designing management of contaminated

2

supplies, improving detection and quantification methods and developing mitigation

strategies (cf. infra 1.1.2.). [4] Mycotoxins may also cause plant diseases. [2]

The Food and Agriculture Organization of the United Nations (FAO) assesses that

circa 25% of the world food crops are polluted with mycotoxins. [5] This is an alarming high

amount especially if the high toxicity is taken into account. Moreover, contamination

imposes high costs on the word’s economy. For these reasons, research have to be done

with regard to the mitigation of mycotoxins.

1.1.2. Mitigation of mycotoxins

Different strategies have been set trying to decrease the amount of mycotoxins. This

mitigation is possible at two levels.

The control of the production of mycotoxins on pre-harvest level is the first approach.

Using Good Agricultural Practices (GAP) is a good way to reduce mycotoxins in food and

feed at pre-harvest level. Examples of GAP are crop rotation, soil management, choice of

variety or hybrid and correct fungicide use. [6, 7]

The second approach is to detoxify contaminated food and feed at post-harvest

level. For this strategy, physical (cleaning and milling processes, thermal processes,

physical adsorption), chemical (by adding ammonia, calcium hydroxide or compounds

which consist of sulfur) and biological methods (malting, brewing and fermentation) can be

used. Different factors as the sort of mycotoxin, the matrix effect, the processing method

and the conditions may influence the degree of mycotoxin reduction. This approach makes

it possible to reduce and/or reuse wastes of low- or non-compliant food batches. However,

most of these approaches seem to be inefficient, unsafe, high-priced and couldn’t be used

at large scale, which is needed for real-world utilization. [5, 8]

3

To improve food safety in future, more investigation needs to be done with regard

to the mitigation of mycotoxins by specific enzymes, e.g. laccases (cf. infra 1.3.). This could

be a promising eco-friendly and affordable method to decrease the amount of mycotoxins

at post-harvest level.

1.2. AFLATOXINS

Aflatoxins are difuranocoumarin derivates, primarily produced by Aspergillus flavus

and A. parasiticus fungi which infect cereal crops including wheat, corn, cotton and nuts.

[9] Dry climate with high temperatures significantly increase the production of these natural

toxins. For these reasons they mainly prevail in West Africa and Southern Europe. With

the trend of the climate change higher temperatures, drought and other conditions for the

production of aflatoxins can be reached more frequently, which is detrimental for the

aflatoxin level in food and feed. [2] So far, over 20 different kind of aflatoxins have been

isolated. The most common types are aflatoxin B1, B2, G1 and G2. [9] For the latter and

aflatoxin M1, there exist maximum levels (see Commission Regulation (EC) No

1881/2006). [10]

1.2.1. Aflatoxin B1

The most predominant mycotoxin is aflatoxin B1 (AFB1) or (6aR,9aS)-2,3,6a,9a-

tetrahydro-4-methoxycyclopenta[c]furo-(3′,2′:4,5)furo[2,3-h][l]benzopyran-1,11-dione. It is

a difuranocoumarin derivate, which contains 5 heterocyclic rings with 6 hydrogen bond

acceptors and no donor hydrogen bonds as can be seen in figure 1.1. [11] This mycotoxin

is classified by the International Agency for Research on Cancer (IARC) as an IARC 1

agent because of the sufficient evidence for being a human carcinogen. [12] Beside the

carcinogenic effect, there is also an immunosuppressive, a hepatotoxic, a mutagenic and

teratogenic effect reported. [11]

4

Figure 1.1. : Chemical structure of aflatoxin B1 [13]

Different metabolites with a broad range of toxicity are generated by the liver trough

cytochrome P450 monooxygenases by reduction, hydroxylation, hydration and epoxidation

reactions. Aflatoxin B1 is mainly metabolized to less toxic metabolites aflatoxin M1 and Q1

and to the major carcinogenic metabolite aflatoxin B1-8,9-epoxide (AFBO) as can been

seen in figure 1.2. The exo-form of AFBO may form adducts with cellular macromolecules,

for example deoxyribonucleic acid (DNA) and proteins, which leads to gene mutations and

cancer. Glutathione on the other hand, binds the AFBO with the help of the enzyme

gluthatione-S-transferase. This avoids the formation of toxic AFBO-adducts. [14, 15]

Figure 1.2. : Overview metabolism aflatoxin B1 [16]

5

1.3. LACCASES

Laccases or p-diphenol dioxygen oxidoreductases are enzymes that belong to the

group ‘blue copper oxidases’. [17] Laccases are able to oxidize a broad range of

substrates, including phenols, diphenols, methoxy-substituted phenols, phenolic, and alkyl

amines. [18]

1.3.1. Characteristics of laccase

The catalytic site of laccase (figure 1.3.a) consists of a cluster of four cupric (Cu2+)

ions, which are ordered in a certain spatial way that each of the magnetic species (T1, T2

and T3) is associated with a polypeptide chain. The Cu-Cu linkages are responsible for the

intense electronic absorption, which give these enzymes a typical blue color (blue copper

oxidases). [17]

For the catalytic cycle (figure 1.3.b), laccase uses one molecular oxygen as electron

acceptor to form radicals resulting in the production of two molecules of water as by-

product. The reduction of molecular oxygen with the release of water takes place at the T2

and T3 sites (trinuclear cluster), while the concomitant oxidation of four substrate

molecules to four substrate radicals seems to take place at the T1 copper site of the

enzyme. Afterwards, the free radicals may undergo coupling reactions hereby producing

dimeric, oligomeric, polymeric, or cross-coupling products. [18]

There are two different scenarios to produce the substrate radicals. The first and

simplest situation is when the substrate molecules directly interact with the copper cluster

for producing the corresponding radicals. Sometimes, substrate molecules don’t have the

capability to do this because they are too large to fit in the active site of the enzyme or

because they have a too high redox potential. These problems can be overcome by adding

6

mediators (cf. infra 1.3.4. ). [18] Until now, it is still uncertain which scenario can be applied

on aflatoxins. [19]

Figure 1.3. : Laccases: active-site structure (a) and catalytic cycle (b) [18]

In nature, these enzymes can be found in higher plants, some insects, some

prokaryotes and almost every fungi. [18] The physiological function depends on the

organism, although they all lead to the catalysis of polymerization or depolymerization

processes. In higher plants (vascular), it is proposed that laccases involve in the cell wall

formation and in the lignification in collaboration with peroxidases. [20] The lignification of

the cell wall leads to a stronger vascular body in higher plants by deposing ill phenolic

polymers (lignins) on the extracellular polysaccharidic matrix. [21] While in white rot fungi,

laccases are mainly responsible for the delignification [20] , which is the removal of lignin

of the extracellular matrix. Furthermore, laccases may be an important virulence factor in

various fungal diseases. [18]

7

1.3.2. Trametes versicolor

During this research laccase, purified from the white rot fungi Trametes versicolor,

was used. Trametes versicolor, also known as Coriolus versicolor is a white rot fungi. The

main function in nature of this group of microorganisms is to enrich the soil via the

degradation of wood. [22] Moreover, this mushroom seems to be good for health and gives

longevity if consumed regularly. [23]

Trametes versicolor produces four laccase isoforms (namely, alfa, beta, gamma and

delta). [1] Because of the multidisciplinary uses of this enzyme (cf. infra 1.3.3.), different

strategies have been set trying to increase the laccases production and productivity by

Trametes versicolor. Adding a mediator as cupper seems to be the most promising method

in order to become this. [24]

1.3.3. Biological applications

Sometimes laccases are called ‘green catalysts’ because they use molecular

oxygen and water is the only by-product they form. This eventually makes them suitable

for biochemical properties or for technological and bioremediation processes. [18]

Laccases are used in food chemistry for the selective removal of phenol derivates

in beverages as wine, beer and fruit juices (stability) and for modifying colours and for the

crosslinking of biopolymers in food. For such applications, the enzymes should be

immobilized because they have not been investigated yet as food additive. Additionally,

laccases may be used in nanobiotechnology (bioelectrochemistry application), in soil

bioremediation, in synthetic chemistry and cosmetics and in the bioremediation of

polyethylene. However, the most widespread application of laccase nowadays is the use

for decolourisation processes of dyes in the textile industry. Another main technological

8

function is the delignification of woody fibers in the pulp and paper industries. [17, 18] In

most of these applications, a chemical mediator is used together with the laccase. (cf. infra

1.3.4.)

1.3.4. Mediators of laccase

As discussed earlier in section 1.3.1., mediators might be added to the substrates

of interest to overcome eventual limitations. Chemical and natural mediators work as

intermediate substrates for laccase. Thus, the mediators are oxidized and the formed

radicals can interact with the bulky or high redox potential substrate hereby widening the

substrate range of laccase. [18]

2,2’-azino-di-(3-ethylbenzotiazoline)-6-sulfonic acid or ABTS is one of the most

famous chemical laccase mediators. The chemical structure of ABTS can be seen in figure

1.4. With a redox potential of 0.67 V, ABTS is likely to be oxidized by the laccases, which

can increase the reactivity further. ABTS has a pKa of 2 and a pH of 3, which makes that

the redox potential is more or less independent of pH. In order to become oxidized, ABTS

needs to behave as a substrate and thus needs to bind to the T1 site of the laccase pocket.

With a Km of order 1-1000 µM, ABTS has a high affinity for laccases compared to many

other substrates. It is known that the different isoforms (α, β, γ or δ) of Trametes versicolor

laccases, have different affinities for the laccases enzymes. This explains the broad range

in Km (1-1000 µM) (cfr. Infra 1.3.5.). [25] During the oxidation of ABTS, the monocation

ABTS+. is formed which is responsible for an intense blue green color (figure 1.4.).

Eventually the dication ABTS2+ is formed by laccases, although there is some

disagreement mentioned in literature. [26]

9

Figure 1.4. : Chemical structure of ABTS [27]

Rutin is a low molecular weight polyphenolic substance, which belongs to the

flavonoids group. Flavonoids can be naturally found in fruits and vegetables. As can been

seen in figure 1.5. rutin hydrate consists of the flavonol quercetin linked to respectively

glucose and rhamnose. As rutin is a polyphenol, it might be a substrate or even a natural

mediator for the laccases enzymes. [28]

Figure 1.5.: Chemical structure of rutin hydrate [27]

10

1.3.5. Preliminary research

In previous research by the food science laboratory group, the interaction of aflatoxin

B1 and aflatoxin M1 with three out of four laccases isoforms of Trametes versicolor (beta,

delta and gamma) was modeled. This made it possible to find differences among the

isoform enzymes in terms of pocket-ligand recognition. [1]

The first step of research consisted of analyzing the sequence of the different

isoforms in order to become their primary structure. The beta isoform of laccase from

Trametes versicolor was already known and was used as template (homology modeling)

for the sequence analysis of the gamma and delta isoforms of Trametes versicolor. Figure

1.6. shows the sequence alignment for the different individual isoforms. Every dot

represents matching residues and dashes or the red color in the blue bar represent gaps.

The binding site of the beta and delta isoforms seemed to be quite similar. The gamma

model was more divergent, due to an extra extend loop. The binding site residues are

indicated in yellow while the green box illustrates the extended region of the gamma

isoform, close to the binding site. [1]

Subsequently, a pharmacophoric analysis was done in order to become a well-

defined structure of the gamma and delta isoforms by using FLAP (Fingerprint for Ligand

And Protein). The pockets seemed to be hydrophobic except for the gamma isoform, which

seemed to be less hydrophobic because of the extra extend loop, which is able to receive

H-bond donor groups. [1]

For estimating the energetic contributions of the binding event between the aflatoxin

and the three different laccase isoforms, the HINT (Hydropathic INTeractions) scoring

function was used. The HINT score is the sum of all inter-atomic contributions from binding,

hereby considering the enthalpy and entropy of the protein-ligand interaction. Thus, the

HINT score may give information about the favor of the host-guest interaction. The higher

11

the score, the higher the favor of the protein-ligand interaction. The results for the aflatoxin

B1 and M1 interaction with the various laccase isoforms can be seen in table 1. [1]

Figure 1.6. : Sequence alignment of the laccase isoforms [1]

Table 1.1. : HINT scores of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) within the

various laccase isoforms. [1]

Laccase isoform

HINT scores

AFB1 AFM1

Beta 248 373

Gamma -199 372

Delta 291 339

12

Table 1.1. shows that aflatoxin B1 may interact with beta (248) and delta (291), but

not with the gamma isoform (negative HINT score). Aflatoxin M1 on the other hand, is

probably able to interact with all the laccase isoforms herein considered. The reason for

the incapability of aflatoxin B1 to interact with the gamma isoform, is probably due to the

fact that aflatoxin B1 isn’t able to sink into the binding site because of the extend loop

present in the gamma isoform. [1]

For the assessment of the procedure reliability of the HINT score docking software,

two benchmark laccase substrates ABTS (cf. supra 1.3.4.) and 2,6-dimethoxyphenol were

used as substrate for the different laccase isoforms. Subsequently, the HINT scores for

these substrate-ligand interactions were compared with experimental research data. At the

end, both results seemed to be comparable, which meant that the HINT score docking

software is reliable for the use of laccase and aflatoxins. [1]

With these results, no conclusion can be made with regard to the degradation

mechanism of aflatoxin B1 or aflatoxin M1 by laccases. Probably the degraded products,

which may be formed for aflatoxin B1 and M1, could be chemically different because of the

different binding architectures in arranging within the laccase catalytic sites. [1]

13

2. OBJECTIVES

As previously mentioned, aflatoxin B1 is known as one of the most carcinogenic

mycotoxins which may be present in food and feed. This is a serious problem for human

health and worldwide economy. Moreover, a lot of the existing mitigation strategies for

aflatoxins seems to be inefficient, unsafe, high-priced or can’t be used at large scale. A

promising and eco-friendly way to battle aflatoxin B1 in future, could be the mitigation of

aflatoxin B1 by laccases enzymes. Preliminary research of the Food science laboratory of

Parma, demonstrated that aflatoxin B1 is able to interact with different strains of laccase

(oxidoreductase enzyme) from Trametes versicolor. Additional experiments need to be

performed in order to investigate this topic more thoroughly.

The first aim of this research was to evaluate the potential of laccases enzymes in

aflatoxin B1 decontamination among various experiments. The simplest in vitro experiment

was performed with aflatoxin B1 in solution, laccases and citrate buffer. During the

treatment time with laccases, the aflatoxin B1 level was monitored and analyzed by the

UHPLC-MS/MS or HPLC-FLD. In other experiments, additional molecules as ABTS

(chemical mediator) and / or rutin hydrate (natural polyphenol), which may trigger the

reaction, were added in order to see if this may change the potential of laccase. Also an

experiment with AFB1 in solution, laccases and a blank maize extract was performed.

It is obvious that beside a decrease of the aflatoxin B1 level, more information about

the mechanism of the enzyme is mandatory. Therefore, the structural elucidation of

degraded products from aflatoxin B1 is necessary. In order to obtain this, the full scan

mode of the Tandem Mass Spectrometry (MS/MS) was used for qualitative mass

spectrometry.

Considering all the data of the various experiments an important question is: Can

we apply the laccases enzymes in real-world on food or feed for reducing the aflatoxin B1

level?

14

3. MATERIAL AND METHODS

3.1. EXPERIMENTAL DESIGN

As discussed in section 1.3.5., preliminary research of the Food science laboratory

of Parma, demonstrated that aflatoxin B1 is able to interact with the catalytic site of different

strains of laccase from Trametes versicolor. These results together with existing literature

provide a basic foothold for this research, wherefore an experimental design had to be

invented.

In line with literature [29], citrate buffer was chosen to set the experiments. It is well

known that the enzyme activity depends on the pH because it may change the enzyme

stability and the electrostatic properties of active site. Therefore some experiments were

performed to choose the appropriate pH. A citrate buffer with a pH of 5.5 was prepared (cf.

infra 3.2.).

Afterwards samples, which consisted of citrate buffer, aflatoxin B1 stock solution

and laccase were prepared. Also controls without laccases enzymes and blanks without

aflatoxin B1 were included. The samples and controls were incubated at 35 °C in tubes for

about 48 hours. To some set of experiments, a chemical mediator (e.g. ABTS) or a natural

compound (e.g. rutin hydrate) were added, believing they could trigger the reaction. For

other samples and controls a blank maize extract was used instead of buffer in order to

see the effect of a natural blank matrix on the enzyme activity (cf. infra 3.3.).

The possible enzymatic reaction was been following during 48 hours. At different

time points (T0, T1 and T3), a specific volume of the incubating Eppendorf tubes was

transferred into another tube. Herein, the enzyme was stopped and removed by following

a procedure with an ethanol/methanol extraction (cf. infra 3.4.). Afterwards the different

samples were analyzed by the UHPLC-MS/MS or HPLC-FLD (cf. infra 3.5.).

15

3.2. MAKING OF CITRATE BUFFER

3.2.1. Chemical agents

Table 3.1. Chemical agents for citrate buffer

Compound Producer Origin

Tri-sodium citrate Fluka Chemie AG Buchs, Switzerland

Citric acid monohydrate SIGMA-ALDRICH Saint Louis, USA

In order to make the citrate buffer, a 0.1 M tri-sodium citrate solution and a 0.1 M

citric acid monohydrate solution were prepared. [30] Therefore, distilled water was used

for making all solvents. The producer and origin of the used compounds can be found in

table 3.1.

3.2.2. Stock solutions of citric acid and sodium tri-acetate – preparation

Both stock solutions were prepared in the same way. 2.1013 g of citric acid was

weighed on the analytical balance (Gibertini, Milan, Italy). Subsequently, the citric acid was

transferred into a beaker and around 30 ml of distilled water was added. A magnetic stir

bar and a magnetic stirrer instrument (FALC instruments, Treviglio, Italy) were used to

make everything homogenous. Afterwards, the solution was carefully transferred into a 100

ml flask. The beaker was rinsed with distilled water and the flask filled until the 100 ml

mark. Finally, the solution was made homogenous by shaking it by hand. [30]

The 0.1 M sodium tri-acetate was prepared in the same way as described above.

Therefore 2.7602 g of sodium tri-acetate was weighed.

16

3.2.3. Combination of stock solutions

Depending on the pH that has to be reached, different volumes of both stock

solutions were combined as can be seen in the table below. [30] 25 ml glass pipets were

used to transfer the volumes of the stock solutions into a flask. Subsequently, the flask was

filled with distilled water until the 100 ml mark. The final step was to verify the pH of the

solution by a pH calculator (Columbus, Ohio, USA).

Table 3.2. Volumes of stock solutions

pH 0.1 M citric acid monohydrate (ml)

0.1 M tri-sodium citrate (ml)

Distilled water (ml)

5.5 16.0 34.0 50.0

3.3. SAMPLE PREPARATION

3.3.1. Chemical agents

Table 3.3. Chemical agents for preparing the samples

Compound Producer Origin

Aflatoxin B1 (AFB1) Biopure Cambridge, USA

Acetonitrile (ACN) SIGMA-ADRICH Saint Louis, USA

Laccase from Trametes versicolor (1.07 U/mg)

SIGMA-ALDRICH Saint Louis, USA

ABTS SIGMA-ALDRICH (FLUKA analytical)

Saint Louis, USA

Rutin hydrate SIGMA-ALDRICH Saint Louis, USA

17

Table 3.3. shows the producer and origin of the different compounds which were

needed for the sample preparation. The 24 ppm aflatoxin B1 stock solution was already

prepared by the Food science laboratory group. The solvent used to dissolve the aflatoxin

was acetonitrile (ACN).

3.3.2. Practical implementation

3.3.2.1. Overview sample list

Table 3.4. shows us the prepared samples, which were made in duplicate except

for the blanks. The controls never contained laccases and were introduced to check if there

are other circumstances, which may change the concentration of aflatoxin B1. Blanks on

the other hand didn’t contain aflatoxin B1 and were added to the sample list in order to

investigate the matrix effect.

Table 3.4. Overview sample list

Sample name

Contains

AFB1

0.5 µM

Citrate

buffer

Laccase

enzyme

ABTS Rutine

hydrate

Maize

extract

AFB1 A and B

AFB1 A and B control

Blank

AFB1 ABTS A and B

AFB1 ABTS A and B control

Blank ABTS

AFB1 rutine A and B

AFB1 rutine A and B control

Blank rutine

AFB1 ABTS rutine A and B

AFB1 ABTS rutine A and B

control

Blank ABTS and rutine

AFB1 maize extract A and B

AFB1 maize extract A and B

control

Blank maize extract

18

3.3.2.2. Aflatoxin B1 samples and controls

In figure 3.1. an overall scheme of the sample preparation is shown. 20 µl of

acetonitrile was added into a 10 ml tube. Subsequently, 16.3 µl of the aflatoxin B1 stock

solution with a concentration of 24 ppm was transferred by a 100.0 µl pipet (Eppendorf

research, Germany) and washed into this tube in order to become an aflatoxin B1

concentration of 0.5 µM. In a next step, this amount was dried under a gentle stream of

nitrogen. An extra amount of acetonitrile was added because the needed volume of

aflatoxin B1 stock solution was very low. Obviously, acetonitrile was used because of its

presence in the aflatoxin B1 stock solution. Moreover it evaporates quite quickly.

20 µl acetonitrile in 10 ml tube

Dry under nitrogen

Add 2.5 ml buffer

Vortex 1 minute

Add laccase enzyme

Vortex 30 seconds

Transfer 500 into Eppendorf tube A and B

Figure 3.1. Overall scheme of the sample preparation

Add aflatoxin B1 stock solution

1) 2)

Transfer 500 µl into

Eppendorf tube A and B

(controls)

Extra

1) Add ABTS

(cf. infra 3.3.3.2.)

2) Add rutin hydrate

(cf. infra 3.3.3.3.)

19

In a following step, the samples were resuspended in 2.5 ml of the citrate buffer (cf.

supra 3.2.) and afterwards they were vortexed for 1 minute. 500 µl was taken in duplicate

from this volume as controls and put into two different 1.5 ml Eppendorf tubes. Thus, the

controls contain citrate buffer with 0,5 µM aflatoxin B1. Subsequently, 0.0105 g of Laccase

enzyme was added (Gibertini, Milan, Italy) to the 10 ml tube in order to become a 7.5 U/ml

solution. Afterwards, the samples were vortexed for 30 seconds to make it homogenous

and finally 500 µl was transferred in duplicate to two new 1.5 ml Eppendorf tubes.

During the experiment the Eppendorf tubes were placed in a warm water bath of

35°C covered with aluminum, because this seemed to be the best conditions for the

possible ongoing reaction. [29] Moreover, each Eppendorf tube contained a magnetic stir

bar. Due to the magnetic stirrer instrument (FALC instruments, Treviglio, Italy) with

temperature control function, a homogenous solution was obtained. Figure 3.2. shows the

instrumental design of the reaction.

Figure 3.2. Instrumental design of the reaction

3.3.2.3. Aflatoxin B1 samples with ABTS and controls

The practical implementation for preparing these samples was identical to the

aflatoxin B1 samples and controls (cf. supra 3.3.2.2.) with the exception of the addition of

ABTS. This molecule was directly added after the addition of aflatoxin B1 stock solution to

20

the 10 ml tube (cf. supra figure 3.1.). 0,0137 g of ABTS was weighed with the analytical

balance (Gibertini, Milan, Italy) in order to become a 10 mM solution in the end. [31]

3.3.2.4. Aflatoxin B1 samples with rutin hydrate and controls

The practical implementation for preparing these samples was identical to the

aflatoxin B1 samples and controls (cf. supra 3.3.2.2.) with the exception of the addition of

rutin hydrate. This molecule was added to the 10 ml tube together with the amount of

aflatoxin B1 stock solution. (cf. supra figure 3.1.). 10 µl of the lower described rutin stock

solution was added by pipet (Eppendorf research, Germany).

The aim was to have a rutin hydrate concentration around 0.5 µM, which is the same

concentration as for aflatoxin B1. A stock solution of rutin hydrate was made, because the

amount needed for the samples was too low to weight. Therefore, 0.010 g was weighed

(Gibertini, Milan, Italy) and dissolved in a small amount of methanol in a beaker.

Subsequently, the volume was transferred into a 100 ml flask. The beaker was rinsed with

methanol and the amount was put into the flask, which was filled until the 100 ml mark.

Finally, the solution was made homogenous by shaking it by hand.

3.3.2.5. Aflatoxin B1 samples with ABTS, rutine hydrate and controls

The practical implementation for preparing these samples was identical to the

aflatoxin B1 samples and controls (cf. supra 3.3.2.2.). In this particular case, 0.0137 g

ABTS and 10 µl of the rutine hydrate stock solution (cf. supra 3.3.2.4.) were added together

with the amount of aflatoxin B1 stock solution (cf. supra figure 3.1.).

21

3.3.2.6. Aflatoxin B1 samples and controls with maize extract

In order to see the effect of the maize extract on the mitigation of aflatoxin B1 by

laccases, experiments were performed in a maize extract instead of citrate buffer.

Therefore 1.25 g of blank maize (without aflatoxins, provided by Italian organization who is

monitoring the occurrence of fumonisins in maize) was put in a 50 ml tube whereat 25 ml

of bi-distilled water was added. During 24 hours the maize extract was placed in a water

bath of 35 degrees with a magnetic stirrer bar inside. Subsequently, the extract supernatant

was passed through cellulose acetate membrane with 0.45 µm pores (CameoTM Filtration

Membrane, Sigma-Aldrich). The obtained extract was stored in refrigerator at 4 °C. [32]

After preparing the maize extract, the samples and controls were made in the same

way as for the preparation of the AFB1 samples and controls (cf. supra 3.3.2.2.) . Therefore

16.3 µl of the aflatoxin B1 stock solution (in order) was put into a 10 ml tube in order to

become 0.5 µM solution, since the maize sample was considered to be free of aflatoxin B1

because no peak for aflatoxin B1 could be detected during HPLC-FLD analysis of the

maize extract. Subsequently, the amount was dried under a gentle stream of nitrogen. After

the addition of 2.5 ml maize extract, the solution was been vortexing during 1 minute. 500

µl was taken in duplicate from this volume as controls and put into two different 1.5 ml

Eppendorf tubes. Thus the controls contain maize extract and aflatoxin B1. Subsequently,

0.0105 g of Laccase enzyme was added (Gibertini, Milan, Italy) to the 10 ml tube in order

to become a 7.5 U/ml solution. Afterwards, the samples were vortexed for 30 seconds to

make it homogenous and finally 500 µl was transferred in duplicate to 2 new 1.5 ml

Eppendorf tubes. The reaction conditions and instrumental design during the ongoing

reaction were the same as for the other samples (cf. supra 3.3.2.2.)

22

3.3.2.7. Blank samples

The different components used for the blank, without aflatoxin B1, can be found in

table 3.4. As can be seen in figure 3.3., the preparation of the blanks is almost simultaneous

to the sample preparation (cf. supra 3.3.3.2.). 0.01752 g of laccases enzyme from

Trametes versicolor was weighed on the analytical balance (Gibertini, Milan, Italy) and

added to the 2.5 ml buffer solution in order to become a solution with an enzyme activity of

7.5 U/ml.

Add rutin hydrate if necessary

Add ABTS if necessary

Dry under nitrogen

Add 2.5 ml buffer

Vortex 1 minute

Add laccase enzyme

Vortex 30 seconds

Transfer 500 µl into Eppendorf tube

Figure 3.3. Overall scheme of the blank preparation

23

3.4. INTERRUPTION OF REACTION - PROCEDURE

3.4.1. Chemical agents

Table 3.5. Chemical agents for interrupting the reaction

Compound Producer Origin

Ethanol Carlo-ERBA reagents Val de Reuil, France

Methanol SIGMA-ALDRICH Saint Louis, USA

An ethanol / methanol solution was made in a proportion of 50/50. Therefore, a

graduated cylinder of 100 ml was used. After this preparation, the solvent was put in a tube

and gently shaken until a homogenous solution was obtained. The producer and origin of

both solvents can be found in table 3.5.

3.4.2. Practical implementation

Immediately after preparing the previous samples (cf. supra 3.3.) on time point T0,

100 µl of each sample, control and blank was taken and transferred into another, new

Eppendorf tube. Herein, 300 µl of ethanol / methanol (50/50) was added. This solution is

able to destruct the structure of proteins like laccase and according to previous

experiments it seemed to be a good way to remove proteins (e.g. serum albumin). [33]

Subsequently, all these Eppendorf tubes were vortexed for 30 seconds and

immediately after placed in crushed ice for at least 10 minutes. Afterwards, the samples

were put in a centrifuge (5810R Eppendorf, Hamburg, Germany) for 15 minutes at a rate

of 10000 rpm (revolutions per minute) and temperature of 4°C. After centrifuging, a pellet

was formed which contained the broken enzymes (proteins). Then, the supernatant was

retrieved from each sample and put in a new Eppendorf tube. Finally, the supernatant

24

sample was put into the freezer, awaiting for analysis. [33] Figure 3.4 shows the extraction

procedure.

This procedure was repeated for other time points (T1 after 24 hours and T2 after

48 hours) during the reaction. The sample on time point T0 (immediately after adding the

enzyme) was important to know the aflatoxin B1 concentration at the beginning of the

reaction.

Transfer 100 µl Eppendorf tube ongoing

reaction in new Eppendorf tube

Add 300 µl ethanol / methanol (50/50)

Vortex 30 seconds

10 minutes in icebath

Centrifuge (4°C, 10.000 rpm, 15 minutes)

Take supernatant

Store in freezer

Figure 3.4. Overall scheme of the extraction procedure for interrupting the reaction

25

3.5. ANALYSIS OF SAMPLES

3.5.1. Chemical agents

Table 3.6. Chemical agent for the analysis of samples

Compound Producer Origin

Acetonitrile SIGMA-ALDRICH Saint Louis, USA

Formic acid SIGMA-ALDRICH Saint Louis, USA

Methanol SIGMA-ALDRICH Saint Louis, USA

The bi-distilled water was obtained after reverse osmosis of water and after purifying

by a Milli-Q plus system ( Millipore, Molsheim, France). For the UHPLC analysis, solution

A was made with bi-distilled water and 0.2 % of formic acid and solution B was prepared

with acetonitrile and 0.2 % formic acid. The formic acid was added in order to obtain a

better ionization. For the HPLC-FLD analysis, solution A consisted of bi-distilled water and

solution B was HPLC-grade methanol.

3.5.2. UHPLC-MS/MS and HPLC with fluorescence detection

Ultra High Performance Liquid Chromatography (UHPLC) (Dionex, Thermo Fisher

Scientific, Germering, Germany) combined with a tandem Mass Spectrometry (MS/MS)

(Triple quadrupole, Thermo Fisher Scientific, San Jose, USA) and a High Performance

Liquid Chromatography (HPLC) (WATERS 2695 separation module, Milford, USA)

combined with a fluorescence detector (FLD) (WATERS 2475 Multi λ fluorescence

detector, Milford, USA) were used to analyze the samples, controls and blanks.

26

3.5.2.1. (U)HPLC

(U)HPLC is an analytical instrument, which makes it possible to separate

components with different polarity and thus different affinity for the specific column. During

this research, an apolar C:18 column (SunShell, ChromaNik Technologies Inc., Japan)

modified with more polar silica groups, was used with measurements 100 mm x 2.1 intern

diameter (i.d.) and particle size 2.6 µM. Due to the extra silica groups, a stronger retention

was obtained, which leaded to a better separation of the various components. The main

difference between an HPLC and UHPLC is that the pump pressure for an UHPLC can go

more up and a smaller particle size of the stationary phase for the UHPLC can be used.

[34]

For analyzing mycotoxins as aflatoxin B1, a reverse phase liquid chromatography

was preferred. This required the use of a polar mobile phase and a more apolar stationary

phase (C:18 modified silica column). In this particular case, a multi-step gradient of mobile

phase was used. As can be seen in table 3.6. and table 3.7., there was a switch from

solvent A (more polar) to solvent B (more apolar) during the run. So, polar components in

the sample were eluted in the beginning of the run and the most apolar components were

eluted later. Due to the multi-step gradient, a better separation and shorter retention times

were become. [34]

Table 3.6. Multi-step gradient for UHPLC

Time Solvent A (aqueous) Solvent B (organic)

1 min 95 % 5 %

11 min 5 % 95 %

16 min 5 % 95 %

17 min 95 % 5 %

25 min 95 % 5 %

27

Table 3.7. Multi-step gradient for HPLC

Time Solvent A (aqueous) Solvent B (organic)

1 min 95 % 5 %

13 min 95 % 5 %

18 min 5 % 95 %

19 min 95 % 5 %

30 min 95 % 5 %

At the start of each run, a flow of 0.350 ml/min of the mobile phase for the UHPLC

(mixture solvent A and B) was obtained. For the HPLC separation a flow of 0.200 ml/min

was set. During the run the flow was kept stable. The samples were injected on the top of

the column and the temperature was set on 40 °C during the run for the UHPLC column

and room temperature was used for the HPLC separation. Both the control of temperature

and pressure were important, especially for avoiding shifts in retention time. The retention

time was about 7.95 min and 16.05 min for respectively the UHPLC and HPLC. After

elution, the components were detected by MS/MS or fluorescence detection.

3.5.2.2. Tandem Mass Spectrometry (MS/MS)

Tandem Mass Spectrometry coupled to the UHPLC was used as detector, which

made it possible to identify and quantify the eluted molecules based on their mass-over-

load ratio (m/z). After eluting, the components were sent through an interface, which is a

small, high vacuum room between the UHPLC and MS/MS. In the interface, the molecule

was ionized due to an electric charge in a positive way (M-H+) or in a negative way

(M-H-). Both for the detection of aflatoxin B1 and rutin hydrate, a positive mode was applied

under high temperature to evaporate the solvent. [35] The whole process is called

‘electrospray ionization’ and can be seen in figure 3.5.

28

Figure 3.5. Overview electrospray ionization (positive) [36]

The formed ions were selected through a triple quadrupole system. A quadrupole

consists of four hyperbolic bars. The first quadrupole was able to isolate the parent ion of

aflatoxin B1 based on its m/z ratio. Subsequently, the parent ions of AFB1 arrived in the

second quadrupole, which is also called the collision cell. There, the parent ions were

collided with a gas, which leaded to the fragmentation of the ions into various daughter

ions depending on the voltage of the collision cell. The third quadrupole served again as a

filter based on its m/z ratio of the specific fragment ions. [35]

Figure 3.6. Overview of triple quadrupole system [36]

29

Subsequently, an electron multiplier converts the ions into an electrical signal which

is detected and proportional to the amount of incoming ions. The Mass Spectral Detector

(MSD) can be used in two different modes, namely the SRM mode (selected-reaction

monitoring) and scan mode.

In the first place, the samples, controls and blanks were run in a SRM mode and

thus they followed the previously described process. Therefore specific fragment ions (with

specific m/z values) of the parental analyte were monitored, which is more sensitive than

the scan mode. After registering the ions, an ion chromatogram was obtained because of

the use of the SRM mode combined with UHPLC. This chromatogram is unique for a

molecule and for this reason it is called a fingerprint of a given molecule.

When there was a statically significant decrease of aflatoxin B1 in a specific set of

experiments, a full scan was performed in order to become a tentative identification of

possible by-products. The use of the (full) scan mode made it possible to registry the m/z

values (ions) beginning from 200 until 900. For using the full-scan mode, the collision cell

(Q2) was shut off and ions were only filtered by Q1 in a broader mass range. In the end, a

mass spectrum with the relative amount of ions in function of the mass-over-load ratio (m/z)

was obtained. The use of scan mode could be useful for the qualitative mass spectrometry.

3.5.2.3. Fluorescence detection (FLD)

The HPLC was coupled to the fluorescence detector (FLD). For using this way of

detection without pre-column derivatization, the analyte of interest should possess native

fluorescence properties. Aflatoxin B1 has fluorescence properties because of the presence

of an aromatic structure. [37]

30

Fluorescence occurs when a compound in the sample absorbs light from the Xenon

lamp with a specific wavelength and hereby comes to a higher energy state (excitation).

When they return to their normal energy state, the absorbed energy is released as photons

(emission). Both the appropriate excitation as emission wavelength are specific for a given

molecule, which makes the fluorescence detection quite selective and sensitive. [37] The

excitation and emission wavelength used for aflatoxin B1 were respectively 365 nm and

425 nm. [38] The fluorescence detector measures the fluorescence emission of the eluted

substances with fluorescence properties. Therefore the emitted signal is amplified by a

photomultiplier. [37]

3.5.3. Statistical analysis

Additionally, to see if the results were statistically significant with 95 certainty an

oneway ANOVA combined with a Tukey POST HOC test was performed for each set of

experiments. As dependent variable the different ratio values of 𝑎𝑟𝑒𝑎 𝑠𝑎𝑚𝑝𝑙𝑒𝑠

𝑎𝑟𝑒𝑎 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑠 were used. As

independent variable, the specific treatment time code (T0, T1 or T2) was used.

31

4. RESULTS

Samples and controls were made in duplicate. To analyze the data of the

experiments, the mean ratio of the aflatoxin B1 area of the samples on the controls (y-axis,

after normalization expressed in percent) was plotted in function of the treatment time (x-

axis). This was the best way to see if the effect is due to the laccase enzyme, while the

effect was adjusted for possible other bias by comparing the samples to the controls. The

standard formulas (1) and (2) were used in order to calculate the mean and the standard

deviation of the results. An overview of the different samples, controls and blanks can be

found in table 3.4. ( cf. supra 3.3.2.).

𝑋 =∑ (𝑛

𝑖 ) 𝑋(𝑖)𝑖=𝑛

𝑖=1

𝑛 With X= mean (%) (1)

∑= sum

X(i)= value ratio 𝑎𝑟𝑒𝑎 𝑠𝑎𝑚𝑝𝑙𝑒𝑠

𝑎𝑟𝑒𝑎 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑠 (%)

n= number of ratios

𝜎 = √1

𝑛∑ (𝑋(𝑖) − 𝑋)2𝑖=𝑛

𝑖=1 With 𝜎= standard deviation (%) (2)

n= number of ratios

∑= sum

X(i) = value ratio 𝑎𝑟𝑒𝑎 𝑠𝑎𝑚𝑝𝑙𝑒𝑠

𝑎𝑟𝑒𝑎 𝑐𝑜𝑛𝑡𝑟𝑜𝑙𝑠 (%)

X= mean (%)

Additionally, to see if the results were statistically significant an oneway ANOVA

(Analysis of variance) combined with a Tukey POST HOC test was performed for each set

of experiments. (cf. supra 3.5.2. Statistical analysis) Moreover the ion ratio was checked

32

for every sample and control to have an idea of the stability of the system. Therefore the

quantifier and qualifier m/z are organized in table 4.1. Using formula (3), the ion ratio

seemed to be stable for every set of experiments.

𝑖𝑜𝑛 𝑟𝑎𝑡𝑖𝑜 = 𝑎𝑟𝑒𝑎 𝑞𝑢𝑎𝑛𝑡𝑖𝑓𝑖𝑒𝑟

𝑎𝑟𝑒𝑎 𝑞𝑢𝑎𝑙𝑖𝑓𝑖𝑒𝑟 (3)

Table 4.1. Quantifier and qualifier values for AFB1 and AFM1

m/z quantifier m/z qualifier

Aflatoxin B1 241.0 285.1

4.1. VARIOUS EXPERIMENTS WITH AFLATOXIN B1

4.1.1. Aflatoxin B1

Figure 4.1. Mean of (area AFB1 samples) / (area AFB1 controls) (%) in function of

the treatment time (h).

0%

20%

40%

60%

80%

100%

120%

140%

t0(0h)

t1(24h)

t2(45h30')M

ean

(are

a sa

mp

les

/ ar

ea c

on

tro

ls)

Treatment time

AFB1 - no mediator

33

Firstly, the area of the AFB1 samples was compared to the area of the controls

which didn’t contain the laccases enzyme. The AFB1 samples and controls were analyzed

by the UHPLC-MS/MS. No real trend for the different ratios in function of the treatment

time can be seen in figure 4.1. Moreover, the statistical oneway ANOVA test shows that

the p value of 0.262 exceeds 0.05 and hereby proves that no trend can be detected with a

certainty of 95 %.

4.1.2. Aflatoxin B1 in presence of ABTS

The analyzing procedure was equally performed as for the AFB1 without mediator

experiment (cfr. supra 4.1.1.). Figure 4.2. shows a reduce for the AFB1 for the samples

compared to the controls in presence of a chemical mediator ABTS and the laccases

enzymes. A decrease of 97.35% (100% - 2.65%) in the AFB1 level for the samples

compared to the controls (without laccases enzyme) was observed in approximately 46 h.

Figure 4.2. Mean of (area AFB1 samples) / (area AFB1 controls) (%) in function of

the treatment time (h).

0%

20%

40%

60%

80%

100%

120%

140%

t0(0h)

t1(24h)

t2(45h30')

Mea

n (a

rea

sam

ple

s /

area

co

ntr

ols

)

Treatment time

AFB1 in presence of ABTS

34

An ANOVA test was performed. The p-value of 0.01 is lower than 0.05 which

suggests that the results are statistically significant with a certainty of 95 %. The additional

Tukey post hoc test shows that the mean of T0 is statistically different from the T1 and T2

time point code. On the other hand, the mean of the T1 group doesn’t statistically differ

from the T2 group.

4.1.3. Aflatoxin B1 in presence of rutin hydrate

Aflatoxin B1 samples and controls in presence of rutine hydrate (0.5 µM) were

injected in the UHPLC-MS/MS. A slight decrease of the level of aflatoxin B1 in the samples

compared to controls can be seen in figure 4.3., although at very low extent. Moreover, this

possible decrease is not statistically significant (p = 0.243 > 0.05).

Figure 4.3. Mean of (area AFB1 samples) / (area AFB1 controls) (%) in function of

the treatment time (h).

The level of rutin hydrate was analyzed during the experiment at different time points

(t0, t1 and t2) by the UHPLC-MS/MS. Surprisingly, the level of rutin hydrate remained

0%

20%

40%

60%

80%

100%

120%

140%

t0(0h)

t1(24h)

t2(45h30')M

ean

(are

a sa

mp

les

/ ar

ea c

on

tro

ls)

Treatment time

AFB1 in presence of rutin hydrate

35

stable during this experiment for the samples compared to the controls as can be seen in

figure 4.4. Thus, it seems that the laccases enzyme doesn’t affect the rutin hydrate, when

using the same concentration as for the AFB1 (0.5 µM).

Figure 4.4. Mean of (area rutin hydrate samples) / (area rutin hydrate controls) (%)

in function of treatment time (h).

4.1.4. Aflatoxin B1 in presence of ABTS and rutin hydrate

These results were obtained after analysis with the UHPLC-MS/MS. As can be

ascertained in figure 4.5., an overall decrease of 93.39% (100% - 6.61%) in the aflatoxin

B1 of the samples compared to the controls was obtained in 46 h. The p-value of 0.02 is

lower than 0.05, which indicates that these results are statistically significant with a

certainty of 95 percent. The Tukey Post Hoc test on the other hand shows that the mean

of T0 (0h) is statistically different from the mean of the T1 (24h) and T2 time point (45h30’),

while the mean of the T1 group doesn’t statistically differ from the mean of the T2 group.

0

0,2

0,4

0,6

0,8

1

1,2

1,4

t0 (0h) t1 (24h) t2(45h30')M

ean

(are

a ru

tin

sam

ples

/ a

rea

ruti

n co

ntro

ls)

Treatment time

Rutin hydrate

36

Figure 4.5. Mean of (area AFB1 samples) / (area AFB1 controls) (%) in function of

the treatment time (h).

Figure 4.6. Mean of (area rutin hydrate samples) / (area rutin hydrate controls) (%)

in function of treatment time (h).

0%

20%

40%

60%

80%

100%

120%

140%

t0(0h)

t1(24h)

t2(45h30')M

ean

(are

a sa

mp

les

/ ar

ea c

on

tro

ls)

Treatment time

AFB1 in presence of ABTS and rutin hydrate

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

t0 (0h) t1 (24h) t2(45h30')

Mea

n (a

rea

ruti

n sa

mpl

es /

are

a ru

tin

cont

rols

)

Treatment time

Rutin hydrate

37

The results for the level of rutin hydrate are plotted in figure 4.6. Also here the level

of rutin hydrate remained more or less stable during this experiment when comparing the

samples to the controls.

4.1.5. Aflatoxin B1 in presence of an natural blank maize extract

Figure 4.7. Mean of (area AFB1 samples) / (area AFB1 controls) (%) in function of

the treatment time (h).

The information about preparation of the maize extract can be found in section

3.3.2.6. The maize extract was used instead of the citrate buffer and no aflatoxin B1 peak

was detected for the extract (blank extract). Hence, AFB1 stock solution was added in

order to obtain a concentration of 0.5 µM. The results are plotted in figure 4.7. and were

analyzed by the HPLC-FLD. A decrease of 51.44% (100% - 48.56%) in the aflatoxin B1

level for samples compared to the controls was obtained in 48 h. This decrease seems to

be statistically significant (p=0.023 < 0.05) with a certainty of 95%.

0%

20%

40%

60%

80%

100%

120%

to (0h) t1 (24h) t2 (48h)Mea

n (a

rea

sam

ple

s /

area

co

ntr

ols

)

Treatment time

AFB1 in presence of a natural blank maize extract

38

4.2. TENTATIVE IDENTIFICATION OF POSSIBLE REACTION PRODUCT(S)

When there was a statistically significant decrease in the level of AFB1, another run

was performed for the same samples, controls and blanks with the UHPLC-MS/MS but in

(full) scan mode instead of SRM mode. This was done in order to become a tentative

identification of possible reaction product(s). Unfortunately, no degradation products in the

full-scan with mass range between 200-900 m/z could be detected when using UHPLC-

MS/MS.

Salt adducts (M-Na+) between aflatoxin B1 and sodium were detected as can be

seen in figure 4.8. Three chromatograms are shown for one sample which contains AFB1,

ABTS and laccase enzyme at the beginning of the reaction (T0). The upper chromatogram

is the ion chromatogram (in SRM mode) for AFB1 (cf. supra 3.5.2.2.). The chromatogram

in the middle represents a reconstructed ion chromatogram (RIC) for the same sample

focused on the m/z 313 (aflatoxin B1, 312 + 1) selected from the Total Ion Chromatogram

(TIC), which was performed in full scan mode. The lowest chromatogram also shows the

RIC focused on the m/z 335 (salt adduct: AFB1 + sodium: 312 + 23) selected from the TIC.

It is clear that the peak at 7.95 min for chromatogram (c) is from AFB1 salt adducts because

aflatoxin B1 has a retention time around 7.97 minutes (Figure 4.8.(a)) which is close to

7.95 min.

The presence of salt adducts with AFB1 implies that not all the aflatoxin B1 is

detected when only focusing on m/z 313 in SRM mode because little amount of the

aflatoxins is hidden because of the salt adducts (m/z 335).

39

Figure 4.8. Relative abundance of ions (%) in function of time (min) with

(a) Ion chromatogram in SRM mode

(b) RIC m/z 313 (AFB1-H+) selected from TIC (full scan)

(c) RIC m/z 335 (AFB1-Na+) selected from TIC (full scan)

a

c

b

40

5. DISCUSSION

5.1. EVALUATION AFLATOXIN B1 DECONTAMINATION POTENTIAL OF

LACCASES

It may be important to understand why there are differences in degradation of

aflatoxin B1 among different experiments and literature. The aflatoxin B1 level didn’t

decrease in the experiment without mediator with the laccases under optimized conditions

(cf. supra 4.1.1.). At first sight, this seems to be not corresponding with the literature. On

the other hand, some of this existing literature didn’t include controls in the proper way,

which are essential for determining the decontamination level of AFB1. Another point that

needs consideration is the fact that some of previous research was performed with

laccases in presence of a natural matrix as culture media. [39] The natural matrix may have

an effect on the enzyme, because low molecular weight components may be present in

this matrix and act as a mediator (cf. supra 4.1.5.). Moreover, during this research pure

fungal laccase (without additional proteins) was used but it is known from literature that

some of the available commercial laccase may contain a mixture of proteins [40], which

are probably able to mediate the enzyme or change the structure of aflatoxin B1.

The addition of a chemical mediator as ABTS for the laccase seems to enhance the

degrading ability of the enzyme. A decrease of 97.35% in aflatoxin B1 was observed after

46h30’ when comparing the samples to the controls (cf. supra 4.1.2.). The results are in

accordance with a similar report, wherein the degradation was approximately 15 % lower.

This is probably due to the lower enzyme activity which was chosen in this experiment and

laccase from Pleurotus pulmonarius was used instead of Trametes versicolor. [31]

However, the addition of ABTS may cost a lot and cause toxicity problems and moreover

a high mediator substrate ratio is needed (cf. infra 5.3.). Therefore natural mediating

compounds are preferred.

41

Rutin hydrate (natural polyphenol) was added in order to see if it may change the

effect of the reaction in the same way as for ABTS. There seems to be a slight decrease

in aflatoxin B1 when comparing the samples to the controls during a period of

approximately 46 h, although it wasn’t statistically significant (cf. supra 4.1.3.). When

adding ABTS and rutin together to the reaction, a statistically significant decrease of

93.39% in aflatoxin B1 was detected for the samples compared to the controls in

approximately 48 h, which is lower than the situation where only ABTS was added to the

reaction. Thus, it seems that the addition of a third molecule, namely rutin hydrate, beside

AFB1 and ABTS may have an negative effect on the reaction. However, the reaction

mechanism is still unknown.

The level of rutin remained stable throughout the reaction in both situations (cf.

supra 4.1.3 and 4.1.4.). This is remarkable since rutin hydrate is a natural flavonoid

polyphenol of which it is supposed to be affected by laccase. On the other hand, not all

phenolic compounds are laccase substrates and can act as mediators. This point may be

important in the context of the possible use in food and feed (cf. infra 5.3.).

Finally an experiment with blank maize extract, AFB1 and laccase (except for

control) was performed (cf. supra 4.1.5.). A statistically significant decrease in AFB1 of

51.44% for the samples compared to the controls was obtained in 48 h. The most likely

explanation for the decrease might be the presence of a natural mediator or a combination

of mediators with eventual synergistic effect in maize, which may trigger the enzymatic

reaction. [32] The overall decrease is lower than when adding ABTS as mediator, although

one needs to be careful with comparing the results of this experiment with other

experiments because of the different analyzing methods which were used (HPLC-FLD

versus UHPLC-MS/MS) and the different reaction substances .

42

5.2. TENTATIVE IDENTIFICATION OF POSSIBLE REACTION PRODUCT(S)

As previously described, there was no detection of reaction products during the

analysis in full scan mode (cf. supra 4.2.). A possible explanation for the missing of

detection of additional peaks from degradation products may be due to the wrong used

mass range, hereby suggesting that dimers, oligomers and polymers together with the

polyphenols or ABTS and aflatoxins could be formed which have an m/z higher than 900.

Besides, it is known that the full scan with UHPLC-MS/MS is not so sensitive. Therefore it

would be interesting to use more sensitive instruments in order to obtain a full scan.

Salt adducts were detected for the samples which were analyzed by UHPLC-MS/MS

as can be seen in figure 4.8. (cf. supra 4.2.) This is due to the citrate buffer, which contains

sodium as counterion. When AFB1 elutes from the UHPLC column, AFB1 is sent through

an interface where the ESI (ElectroSpray Ionization) in positive mode of aflatoxin B1 takes

place. In the interface sodium from the citrate buffer may be present when the AFB1 arrives

in the interface. This may cause the creation of sodium-AFB1 adducts, which are not

detected in the ion chromatogram focused on m/z 313 (SRM mode). It may occur that the

formation of sodium adducts is not so stable, which may declare the fluctuations in areas.

Moreover the presence of sodium may change the adducts. This analysis bias is an

analytical problem and can change according to the temperature, the sodium

concentration, the applied cone voltages… [41]

As previously mentioned, it would be interesting to use more sensitive devices in

order to perform a chromatogram in full scan in order to detect possible degradation

products. Therefore, it is important to remove the sodium before analyzing the samples

again because the sodium may change the possible formed degradation products during

the analysis which would make the interpretation quite challenging. Moreover, one needs

to keep in mind that the addition of mediators may change the possible products. Analysis

43

of the samples by Q-TOF-Mass Spectrometer (Quadrupole Time-Of-Flight mass

spectrometer) could be promising because of the high sensitivity structural elucidation tool.

5.3. POSSIBLE REAL-WORLD APPLICATION IN FOOD AND FEED

The various data, didn’t give definite information about the mechanism of the

enzyme although it is important to know the reaction mechanism before applying this

mitigation strategy on food or feed for consumption. Furthermore, knowledge of the

mechanism will make the optimization of the reaction conditions possible, which is

necessary for upscaling this strategy for real-world applications. In order to obtain

information about the reaction mechanism, the identification of degradation products of

AFB1 is mandatory (cf. supra 5.2.). Also the toxicity and stability of possible reaction

products has to be further determined and evaluated in future. The formation of more toxic

by-products must be avoided. However, a decreased genotoxicity was already reported in

literature for AFB1 after treatment with laccase. [29]

It is difficult to make conclusions about the possible application of laccases in real-

world because none of the above described data are available at this moment. Data from

this research demonstrates that he use of laccase could be a promising way to decrease

the AFB1 level, but only if a mediating system is present and only if further investigation

shows that the degradation products are not toxic and stable during time. Furthermore, one

needs to keep in mind that the use of a chemical mediator like ABTS can’t be used for food

and feed applications because of its toxicity. [31] Therefore, it is clear that natural occurring

mediators need to be used. A natural mediator for laccase could be present in the food or

feed matrix [32] as it probably can be found in the used maize extract of the experiment

(cf. infra 4.1.5.).

Beside mediators also other phenolic substances, which can be substrates for

laccase, may be present in the maize extract. These substances of the food or feed matrix

44

may be changed or oxidized by the laccase, which may have negative consequences for

the nutrition value of food and feed. This must be avoided. On the other hand, the level of

rutin hydrate (phenolic substance, 0.5 µM) remained stable in presence of the laccase

enzyme, AFB1 (0.5 µM) and ABTS (cf. supra Figure 4.6.). This demonstrates that laccase

can be quite specific for aflatoxin B1 and thus may support the potential use for food or

feed applications.

45

6. CONCLUSION

The aflatoxin B1 level didn’t decrease in presence of laccase in the experiment

without mediator (cf. 4.1.1.). A statistically significant reduction of the AFB1 level caused

by laccase, has only been observed if a mediating system was present. The most

spectacular reduction in AFB1 was obtained when a chemical mediator (ABTS) was added

to the reaction. Although, one needs to keep in mind that ABTS can’t be used in food or

feed because of its toxicity.

Removal of 97.37% and 93.39% of AFB1 by laccase was achieved in 46 hours using

ABTS or ABTS together with rutin hydrate. Remarkable was the fact that the level of

polyphenol rutin hydrate remained stable in presence of enzyme mediator ABTS, AFB1

and laccase, which supports that laccase might be quite specific for aflatoxin B1. This

eventually supports a potential for using laccase in food and feed. For the samples with

maize extract, a decrease of 51.44 % was observed in 48 hours which may suggest that

natural compounds or even a natural mediator(s) may be present in the maize extract

which can trigger the enzymatic reaction.

Unfortunately no reaction product(s) were detected when using the UHPLC-MS/MS

in full scan mode. Although the detection of degradation products is necessary in order to

gain more information about the enzyme mechanism.

It is quite difficult to make conclusions about the possible application of laccases in

real-world for food and feed because of the lack of information. Considering all the previous

data, the use of laccase (from Trametes versicolor) seems to be an effective, green way

and maybe quite specific tool for reducing the aflatoxin B1 level in food and feed. However,

further studies are required in order to identify reaction product(s) of AFB1 by using a more

sensitive detector (e.g. Q-TOF-Mass Spectrometer), evaluate toxicity and stability of

reaction product(s). Knowledge of the enzyme mechanism for aflatoxin B1, will be

46

necessary for an eventual upscale of this mitigation strategy in future. In addition, testing

the influence of laccases on different food or feed products naturally contaminated with

aflatoxin B1, would be interesting in order to evaluate the food quality, its food safety and

the potential of laccase in food and feed after the mitigation.

47

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