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
67
Embed
MITIGATION OF AFLATOXIN B1 BY LACCASES FROM TRAMETES ... · statistisch significante daling in aflatoxine B1 waargenomen door laccase. Een daling van 97.37% en 93.39% voor respectievelijk
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
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.
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
7. REFERENCES
1. Dellafiora, L., et al., Degradation of Aflatoxins by Means of Laccases from Trametes versicolor: An In Silico Insight. Toxins, 2017. 9(1).
2. Marroquin-Cardona, A.G., et al., Mycotoxins in a changing global environment - A review. Food and Chemical Toxicology, 2014. 69: p. 220-230.
3. EURL. Legislation on mycotoxins. 07/06/2017 [cited 2017 20/03/2017]; Available from: https://ec.europa.eu/jrc/en/eurl/mycotoxins/legislation.
4. Aswani Kumar YVV1, R.R., Bodaiah B1, Usha Kiranmayi Mangamu2, Vijayalakshmi M2 and Sudhakar Poda1* Mycotoxin Strategies: Impact on Global Health and Wealth. Pharmaceutica Analytica Acta, 2016.
5. Chilaka, C.A., et al., The Status of Fusarium Mycotoxins in Sub-Saharan Africa: A Review of Emerging Trends and Post-Harvest Mitigation Strategies towards Food Control. Toxins, 2017. 9(1).
6. He, J.W., et al., Chemical and biological transformations for detoxification of trichothecene mycotoxins in human and animal food chains: a review. Trends in Food Science & Technology, 2010. 21(2): p. 67-76.
7. Commission recommendation on the prevention and reduction of Fusarium toxins in cereals and cereal products., E. commission, Editor. 2006: Brussels.
8. Zhu, Y., et al., Innovative technologies for the mitigation of mycotoxins in animal feed and ingredients-A review of recent patents. Animal Feed Science and Technology, 2016. 216: p. 19-29.
9. Kumar, P., et al., Aflatoxins: A Global Concern for Food Safety, Human Health and Their Management. Frontiers in Microbiology, 2017. 7.
10. Commission, E. Aflatoxins. Available from: https://ec.europa.eu/food/safety/chemical_safety/contaminants/catalogue/aflatoxins_en.
11. NCBI, P. Aflatoxin B1. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/aflatoxin_b1#section=Top.
12. IARC Aflatoxins. 225-248.
13. Sigma-Aldrich.
14. NCBI, P. Aflatoxin M1. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/aflatoxin_m1#section=Top.
15. Murphy, P.A.H., S. ; Landgren,C. ; Bryant, C.M., Food mycotoxins: An update. Journal of Food Science, 2006. 71: p. R51 - R65.
16. Rawal, S. and R.A. Coulombe, Metabolism of aflatoxin B-1 in Turkey liver microsomes: The relative roles of cytochromes P450 1A5 and 3A37. Toxicology and Applied Pharmacology, 2011. 254(3): p. 349-354.
17. Gomaa, O.M. and O.A. Momtaz, Copper induction and differential expression of laccase in Aspergillus flavus. Brazilian Journal of Microbiology, 2015. 46(1): p. 285-292.
18. Riva, S., Laccases: blue enzymes for green chemistry. Febs Journal, 2013. 280: p. 590-590.
19. Alberts, J.F., et al., Degradation of aflatoxin B(1) by fungal laccase enzymes. Int J Food Microbiol, 2009. 135(1): p. 47-52.
20. Mayer, A.M. and R.C. Staples, Laccase: new functions for an old enzyme. Phytochemistry, 2002. 60(6): p. 551-565.
21. Ros Barcelo, A., Lignification in plant cell walls. Int Rev Cytol, 1997. 176: p. 87-132.
22. Shah, V., et al., Influence of iron and copper nanoparticle powder on the production of lignocellulose degrading enzymes in the fungus Trametes versicolor. Journal of Hazardous Materials, 2010. 178(1-3): p. 1141-1145.
23. Wang, F.F., et al., A Review of Research on Polysaccharide from Coriolus versicolor, in Proceedings of the 2012 International Conference on Applied Biotechnology, T.C. Zhang, et al., Editors. 2014. p. 393-399.
24. Lorenzo, M., D. Moldes, and M.A. Sanroman, Effect of heavy metals on the production of several laccase isoenzymes by Trametes versicolor and on their ability to decolourise dyes. Chemosphere, 2006. 63(6): p. 912-7.
25. Christensen, N.J. and K.P. Kepp, Setting the stage for electron transfer: Molecular basis of ABTS-binding to four laccases from Trametes versicolor at variable pH and protein oxidation state. Journal of Molecular Catalysis B-Enzymatic, 2014. 100: p. 68-77.
26. Munteanu, F.D., et al., Staining of wool using the reaction products of ABTS oxidation by Laccase: Synergetic effects of ultrasound and cyclic voltammetry. Ultrasonics Sonochemistry, 2007. 14(3): p. 363-367.
27. Sigma-Aldrich. Available from: http://www.sigmaaldrich.com.
28. Hollman, P.C.H. and I.C.W. Arts, Flavonols, flavones and flavanols - nature, occurrence and dietary burden. Journal of the Science of Food and Agriculture, 2000. 80(7): p. 1081-1093.
29. Zeinvand-Lorestani, H., et al., Comparative study of in vitro prooxidative properties and genotoxicity induced by aflatoxin B1 and its laccase-mediated detoxification products. Chemosphere, 2015. 135: p. 1-6.
30. Gomori, G. Preparation of Buffers for use in Enzyme Studies. 2004 14/06/2004 13/04/2017]; Available from: https://webcache.googleusercontent.com/search?q=cache:g02Bwbi_1XwJ:https://www.researchgate.net/file.PostFileLoader.html%3Fid%3D5726eb24dc332d1e7743a98a%26assetKey%3DAS%253A357174124531720%25401462168356006+&cd=6&hl=nl&ct=clnk&gl=be.
31. Loi, M., et al., Aflatoxin B(1) and M(1) Degradation by Lac2 from Pleurotus pulmonarius and Redox Mediators. Toxins (Basel), 2016. 8(9).
32. Liang, S., Q. Luo, and Q. Huang, Degradation of sulfadimethoxine catalyzed by laccase with soybean meal extract as natural mediator: Mechanism and reaction pathway. Chemosphere, 2017. 181: p. 320-327.
33. Dellafiora, L., et al., Assessing the hydrolytic fate of the masked mycotoxin zearalenone-14-glucoside - A warning light for the need to look at the "maskedome". Food and Chemical Toxicology, 2017. 99: p. 9-16.
35. McLafferty, F., Tandem mass spectrometry. Science, 1981. 214(4518): p. 220_287.
36. Available from: https://www.hindawi.com/journals/ijac/2012/282574/fig6/s. (16/03/2017).
37. Waters. 2475 Multi Fluorescence Detector - operator's guide. 2012; Available from: https://www.waters.com/webassets/cms/support/docs/715003812ra_2475_multiwave_flr_detector_ops_guide.pdf.
38. Huang, J.E., D. Analysis of Aflatoxins Using Fluorescence Detection. 2007; Available from: http://tools.thermofisher.com/content/sfs/brochures/App-Note-381-Analysis-of-Aflatoxins-Using-Fluorescence-Detection.pdf.
39. Kachouri, F., H. Ksontini, and M. Hamdi, Removal of Aflatoxin B1 and Inhibition of Aspergillus flavus Growth by the Use of Lactobacillus plantarum on Olives. Journal of Food Protection, 2014. 77(10): p. 1760-1767.
40. Margot, J., et al., Bacterial versus fungal laccase: potential for micropollutant degradation. Amb Express, 2013. 3.
41. Kruve, A. and K. Kaupmees, Adduct Formation in ESI/MS by Mobile Phase Additives. Journal of the American Society for Mass Spectrometry, 2017. 28(5): p. 887-894.