Ochratoxin A adsorption phenotype: an inheritable yeast trait

(1)

Andrea CARIDI

1 , Rossana SIDARI

1 , Andrea PULVIRENTI

2 , Giuseppe MECA

3 , Alberto RITIENI

3

1

Unit of Microbiology, Department of Scienze e Tecnologie Agro-Forestali e Ambientali (DiSTAfA), "Mediterranea"

University of Reggio Calabria, Via Feo di Vito, I-89122 Reggio Calabria, Italy

2

Department of Scienze Agrarie e degli Alimenti, University of Modena e Reggio Emilia, Via J.F. Kennedy 17, I-42100

Reggio Emilia, Italy

3

Department of Scienza degli Alimenti, “Federico II” University of Napoli, Parco Gussone ed. 84, I-80055 Portici

(NA), Italy

OCHRATOXIN A ADSORPTION PHENOTYPE: AN INHERITABLE YEAST TRAIT

Ochratoxin A (OTA,) bio-produced by a few species belonging to Aspergillus and Penicillium genera, is a very dangerous secondary metabolite, frequently noticed in various foods and beverages. Since 1996, OTA has been reported in grapes, grape juices and wines (1). Different decontamination procedures based on

Saccharomyces strains were recently proposed for OTA removal (2-4). Interestingly the OTA decrease is strain-dependent (5). Remarkable differences among wine

yeasts - both Saccharomyces (6) and non-Saccharomyces (7) - have been reported in the OTA sequestering activity during winemaking; this may depend, among other factors, on the different mannosylphosphate content in the mannoproteins of wine yeasts. At present, no study has been carried out to analyse the inheritable nature of the “OTA adsorption” phenotype.

AIM OF THE WORK

This work aims to evaluate the inheritance of the trait “OTA adsorption” during winemaking, testing a population of 46 descendants and their two parental wine strains of Saccharomyces cerevisiae.

INTRODUCTION

REFERENCES

1. Zimmerli, B., Dick, R., 1996. Ochratoxin A in table wine and grape-juice: occurrence and risk assessment. Food Additives and Contaminants 13, 655–668.

2. Piotrowska, M., Zakowska, Z., 2000. The biodegradation of ochratoxin A in food products by lactic acid bacteria and baker’s yeast. Food Biotechnology 17, 307–310.

3. Bejaoui, H., Mathieu, F., Taillandier, P., Lebrihi, A., 2004. Ochratoxin A removal in synthetic and natural grape juices by selected oenological Saccharomyces strains. Journal of Applied Microbiology 97, 1038– 1044.

4. Caridi, A., Galvano, F., Tafuri, A., Ritieni, A., 2006a. In-vitro screening of Saccharomyces strains for ochratoxin A removal from liquid medium. Annals of Microbiology 56, 135–137.

5. Scott, P.M., Kanhere, S.R., Lawrence, G.A., Daley, E.F., Farber, J.M., 1995. Fermentation of wort containing added ochratoxin A and fumonisins B1 and B2. Food Additives and Contaminants 12, 31–40. 6. Caridi, A., Galvano, F., Tafuri, A., Ritieni, A., 2006b. Ochratoxin A removal during winemaking. Enzyme and Microbial Technology 40, 122–126.

7. Cecchini, F., Morassut, M., Garcia Moruno, E., Di Stefano, R., 2006. Influence of yeast strain on ochratoxin A content during fermentation of white and red must. Food Microbiology 23, 411–417. 8. Caridi, A., Crucitti, P., Ramondino, D., 1999. Winemaking of musts at high osmotic strength by thermotolerant yeasts. Biotechnology Letters 21, 617–620.

MATERIALS AND METHODS

Two parental wine strains of Saccharomyces cerevisiae named TP5 and TT173, and 46 single-spore cultures were used. The parental strains had been isolated from native microflora of wine fermentations, selected for oenology (8), and strain TT173 had also been tested for OTA removing capacity in saline solution (4) and during winemaking (6). The 46 single-spore cultures – 32 obtained from the strain TP5 and 14 from the strain TT173 – were isolated by micromanipulator.

Each strain was inoculated (1 ml) in triplicate in test tubes containing 10 mL of must obtained from the Calabrian Zibibbo white grape variety artificially contaminated with OTA to reach a total content of 4.10 ng/mL. The microvinification trials were performed at 25°C; after 30 days, the OTA analysis in wines (residual OTA), in the saline solution used to wash lees (adsorbed OTA on cell wall), and in the lees after the washing (linked OTA) were carried out.

The determination of OTA content in wines was performed by HPLC using LC-10AD pumps and the RF-10Axl (Shimadzu, Japan) fluorescence detector. Data acquisition and handling were made by a system control SLC10A with software VP5 (Shimadzu, Japan). A Synergi (Phenomenex, USA) C18 (250mm×4.6 mm,

5μm) column was used. The HPLC conditions were: constant flow of 1 mL/min and CH₃CN (1% acetic

acid)-H₂O (1% acetic acid) (50:50 v/v) as the starting eluent system. The starting ratio was linearly modified to

100% CH₃CN in 15 min. From the 15th _{to 18}th _{min the pumps were taken back to starting conditions and}

then the isocratic conditions were taken for 5 min. Samples were filtered through a 0.22μm syringe filter (Millipore, Bedford, MA, USA) prior to injection (20μL) into the HPLC column by 250mL syringe (Hamilton, Switzerland).

The OTA adsorbed on yeast cell walls was determined as follows: cells were harvested by centrifugation (centrifuge Juan model CR3i) and washed twice (4000 rpm for 10 min at 4 °C) with saline solution. The two aliquots of saline solution were combined and analysed as above described for wines.

The OTA content in lees after the washing was determined as follows: cells were harvested by centrifugation, sonicated in the saline solution for 30 min and washed twice (4000 rpm for 10 min at 4 °C) with saline solution. The pellet was suspended in 3mL of saline solution, 3mL of ethyl acetate were added and, after mixing and centrifugation (4000 rpm for 10 min at 4 °C), 1mL of the top phase was evaporated by Rotavapor (system Juan model RC60), re-suspended in 1mL of methanol and analysed by injecting 20μL into the HPLC column by 100μL syringe (Hamilton, Switzerland).

All the analytical data were subjected to statistical analysis using StatGraphics Centurion XV for Windows XP from StatPoint.

RESULTS

The residual OTA in wines varied from 0.74 to 3.18 ng/mL; the OTA adsorbed on yeast cell walls varied from 0.60 to 2.95 ng/mL while the OTA linked to the lees varied from 0.01 to 2.69 ng/mL.

The majority of the descendants exhibited significant (P<0.05) differences from their parental strains (Table 1); different distribution patterns of residual OTA in wines, adsorbed OTA on cell walls, and OTA linked to cells were shown for each parental strain.

OTA residual in wine: strain TT173 showed a majority of descendants with significantly different values,

while strain TP5 had the least number of descendants with significantly different values.

OTA adsorbed on cell walls: strain TP5 exhibited for all the progeny significantly different values

compared to the parental strain, strain TT173 showed a majority of descendants with significantly different values.

OTA linked to cells: strain TT173 exhibited for the overwhelming majority of the progeny significantly

different values compared to the parental strain, while strain TP5 showed less than 50% of descendants with significantly different values.

Figures show the respective positions of parental strains within the descendant distribution were different both among parameters and parental strains. Data represent the mean of three replicates; standard deviation is reported on the bar graph.

OTA residual in wines: the parental strains TP5 and TT173 were, respectively, the 22nd _{out 33 (Fig. 1)}

and the 5th _{out 15 (Fig. 2) among their progeny.}

OTA adsorbed on cell walls: the two wine yeasts were positioned 32nd _{(Fig. 3) and 12}th _{(Fig. 4).}

OTA linked to cells: the parental strains were, respectively, the 20th _{(Fig. 5) and the 6}th _{(Fig. 6) among}

their progeny.

It is interesting to note that progeny deriving from the same ascus occasionally were included in the same homogeneous group according to Least Significant Difference analysis (P < 0.05), such as for the three single-spore cultures deriving from the ascus 9 - TP5 progeny - regarding the residual OTA in wine (Fig.

1). This phenomenon may be exploited by trying to cross these neighbouring progenies in order to

strengthen the strain traits. Concerning progeny from the same ascus, significant differences were observed among the TP5 descendants for the asci 1, 2, 7, 12 and among the TT173 descendants for asci 1, 4, 5, 6 (Fig. 1-2), among the TP5 descendants for the asci 1, 2, 5, 8, 10, 11, 12 and among the TT173 descendants for asci 1, 4, 6 (Fig. 3-4) and among the TP5 descendants for the asci 1, 3, 5, 7, 8, 10, 11, 12 and among the TT173 descendants for asci 1, 3, 4, 6 (Fig. 5-6), respectively for residual OTA in wine, adsorbed OTA on cell wall and OTA linked to cell.

Results demonstrated that the “OTA adsorption” trait segregates through the progeny showing amplitude of distribution that is peculiar for each strain.

PERSPECTIVES

The analysis of the progeny carried out with this study demonstrated that the “OTA adsorption” is genetically controlled, therefore it is an inheritable trait of wine yeasts.

It is interesting to note that the majority of the descendants are characterised by a great and significant diversity compared to their parents. These findings are the basis of a future work to demonstrate the mechanisms of inheritance, thus they constitute an initial step for establishing breeding strategies in order to improve wine yeast as regards the “OTA adsorption” trait.

This will make it possible to drastically reduce,

during winemaking, the OTA content of

contaminated musts using wine yeasts improved by genomic strategies.

Table 1 - “Ochratoxin A adsorption” phenotype of two wine strains and their progenies studied by microvinification trials.

TP5 TT173

Progeny Progeny

Parameters Parent Mean Range %* Parent Mean Range %*

Residual OTA in wines 1.42 1.35 0.74-1.86 15.62 1.60 1.81 1.32-3.18 57.14

Adsorbed OTA on cell walls 2.64 0.96 0.60-2.95 100.00 1.14 0.98 0.72-1.75 78.57

OTA linked to cells 0.12 0.28 0.01-2.69 46.87 0.17 0.74 0.06-2.07 85.71

1

* Percentage of descendants included in homogeneous groups (P < 0.05 according to Least

Significant Difference analysis) that do not include their parental strain.

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Figure 1 - Distribution of the parameter “residual OTA in wine” for strain TP5 and its derived spore clones.

The OTA value is expressed as ng/mL. Bar graphs indicate residual OTA in wine obtained by the parental strain (black area) and the derived spore clones (hatched area). Progeny deriving from the same ascus is shown with the same hatching.

h g f f e de c d e c d e cd c bc b b a a 1.0 1.5 2.0 2.5 3.0 3.5

Figure 2 - Distribution of the parameter “residual OTA in wine” for strain TT173 and its derived spore clones.

n m l l k jk ijk h ijk h ijk g h ijk fg h ij e fg h ij e fg h i d e fg h c d e fg h c d e fg h c d e fg h c d e fg h c d e fg c d e fg h b c d e fg b c d e f b c d e f b c d e f b c d e f b c d e f b c d e b c d e b c d e b c d abc ab a 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Figure 3 - Distribution of the parameter “adsorbed OTA on cell wall” for strain TP5 and its derived spore

clones. The OTA value is expressed as ng/mL. Bar graphs indicate residual OTA in wine obtained by the parental strain (black area) and the derived spore clones (hatched area). Progeny deriving from the same ascus is shown with the same hatching.

f e de cd bc bc b ab ab ab a a a a a 0.0 0.5 1.0 1.5 2.0

Figure 4 - Distribution of the parameter “adsorbed OTA on cell wall” for strain TT173 and its derived spore

clones. The OTA value is expressed as ng/mL. Bar graphs indicate residual OTA in wine obtained by the parental strain (black area) and the derived spore clones (hatched area). Progeny deriving from the same ascus is shown with the same following hatching.

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Figure 5 - Distribution of the parameter “OTA linked to cell” for strain TP5 and its derived spore clones.

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2.5 Figure 6 - Distribution of the parameter “OTA

linked to cell” for strain TT173 and its derived spore clones. The OTA value is expressed as ng/mL. Bar graphs indicate residual OTA in wine obtained by the parental strain (black area) and the derived spore clones (hatched area). Progeny deriving from the same ascus is shown with the same following hatching. 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8

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bcd

e

bcd

e

bcd

e

bcd

abc

ab

a

0.0 1.0 2.0

m

lm

klm

jklm

ijkl

m

hijk

lm

hijk

lm

ghi

jklm

fgh

ijkl

efg

hijk

l

efg

hijk

l

def

ghi

jk

def

ghi

jk

cde

fgh

ijk

cde

fgh

ijk

cde

fgh

ijk

cde

fgh

ijk

cde

fgh

ij

cde

fgh

il

cde

fgh

ij

cde

fgh

i

bcd

efg

h

bcd

efg

bcd

efg

bcd

ef

bcd

e

bcd

e

bcd

e

bcd

abc

ab

a

0.0

1.0

2.0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefg

h

cdefgh

i

cdefgh

ij

cdefgh

il

cdefgh

ij

cdefgh

ijk

cdefgh

ijk

cdefgh

ijk

cdefgh

ijk

defghij

k

defghij

k

efghijk

l

efghijk

l

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

g

_bcdef

g

bcdef

gh

cdefg

hi

cdefg

hij

cdefg

hil

cdefg

hij

hijk

cdefg

hijk

cdefg

hijk

cdefg

hijk

defgh

ijk

defgh

ijk

l

efghijk

l

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0

2,0 T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefgh

_cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefgh

cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefgh

_cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefgh

cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

g

_bcdef

g

bcdef

gh

cdefg

hi

cdefg

hij

cdefg

hil

cdefg

hij

hijk

cdefg

hijk

cdefg

hijk

cdefg

hijk

defgh

ijk

defgh

ijk

l

efghijk

l

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefgh

_cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0

1,0 2,0

T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefgh

_cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

m

lm

klm

jklm

ijklm

hijklm

ghijk

lm

fghijk

l

efgh

ijkl

efgh

ijkl

defg

hijk

defg

hijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghij

cdef

ghil

cdef

ghij

cdef

ghi

bcde

fgh

bcde

fg

bcde

fg

bcde

f

bcde

bcd

abc

ab

a

0.0 1.0 2.0

m

lm

klm

jklm

ijkl

m

hijk

lm

hijk

lm

gh

ijkl

m

fgh

ijkl

efg

hijk

l

efg

hijk

l

de

fgh

ijk

de

fgh

ijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hij

cd

efg

hil

cd

efg

hij

cd

efg

hi

bc

de

fgh

bc

de

fg

bc

de

fg

bc

de

f

bc

de

bc

de

bc

de

bc

d

bc

d

abc

ab

a

0.0

1.0

2.0 m

lm

kl

m

jk

lm

ijk

lm

hi

jk

lm

hi

jk

lm

gh

ijk

lm

fg

hi

jk

l

ef

gh

ijk

l

ef

gh

ijk

l

de

fg

hi

jk

de

fg

hi

jk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ij

cd

ef

gh

il

cd

ef

gh

ij

cd

ef

gh

i

bc

de

fg

h

bc

de

fg

bc

de

fg

bc

de

f

bc

de

bc

de

bc

de

bc

d

bc

d

abc

ab

a

0.0

1.0

2.0

T P5

a

_ab

_abc

_bcd

_bcde

f

_bcde

fg

_bcde

fg

bcde

fgh

cdef

ghi

cdef

ghij

cdef

ghil

cdef

ghij

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

fghijk

l

ghijk

lm

hijklm

ijklm

jklm

klm

lm

m

0,0

1,0 2,0

TP5

a

_ab

_abc

_bcd

e

_bcd

e

_bcd

e

_bcd

ef

_bcd

efg

_bcd

efg

bcd

efgh

cde

fghi

cde

fghi

j

cde

fghi

l

cde

fghi

j

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

_fghi

jkl

ghijk

lm

hijkl

m

hijkl

m

_ijklm

_jklm

_klm

_lm

_m

0,0 1,0 2,0 T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

g

_bcdef

g

bcdef

gh

_cdefg

hi

cdefg

hij

cdefg

hil

cdefg

hij

hijk

cdefg

hijk

cdefg

hijk

cdefg

hijk

defgh

ijk

defgh

ijk

l

efghijk

l

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

TP5

a

_ab

_abc

_bcd

e

_bcd

e

_bcd

e

_bcd

ef

_bcd

efg

_bcd

efg

bcd

efgh

cde

fghi

cde

fghi

j

cde

fghi

l

cde

fghi

j

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

_fghi

jkl

ghijk

lm

hijkl

m

hijkl

m

_ijklm

_jklm

_klm

_lm

_m

0,0 1,0 2,0 T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

bcdefg

bcdefgh

cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

m

lm

klm

jklm

ijklm

hijklm

ghijk

lm

fghijk

l

efgh

ijkl

efgh

ijkl

defg

hijk

defg

hijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghij

cdef

ghil

cdef

ghij

cdef

ghi

bcde

fgh

bcde

fg

bcde

fg

bcde

f

bcde

bcd

abc

ab

a

0.0 1.0 2.0

m

lm

klm

jklm

ijkl

m

hijk

lm

hijk

lm

gh

ijkl

m

fgh

ijkl

efg

hijk

l

efg

hijk

l

de

fgh

ijk

de

fgh

ijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hij

cd

efg

hil

cd

efg

hij

cd

efg

hi

bc

de

fgh

bc

de

fg

bc

de

fg

bc

de

f

bc

de

bc

de

bc

de

bc

d

bc

d

abc

ab

a

0.0 1.0 2.0

m

lm

kl

m

jk

lm

ijk

lm

hi

jk

lm

hi

jk

lm

gh

ijk

lm

fg

hi

jk

l

ef

gh

ijk

l

ef

gh

ijk

l

de

fg

hi

jk

de

fg

hi

jk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ij

cd

ef

gh

il

cd

ef

gh

ij

cd

ef

gh

i

bc

de

fg

h

bc

de

fg

bc

de

fg

bc

de

f

bc

de

bc

de

bc

de

bc

d

bc

d

abc

ab

a

0.0

1.0

2.0

T P5

a

_ab

_abc

_bcd

_bcde

f

_bcde

fg

_bcde

fg

bcde

fgh

cdef

ghi

cdef

ghij

cdef

ghil

cdef

ghij

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

fghijk

l

ghijk

lm

hijklm

ijklm

jklm

klm

lm

m

0,0

1,0 2,0

TP5

a

_ab

_abc

_bcd

e

_bcd

e

_bcd

e

_bcd

ef

_bcd

efg

_bcd

efg

bcd

efgh

cde

fghi

cde

fghi

j

cde

fghi

l

cde

fghi

j

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

_fghi

jkl

ghijk

lm

hijkl

m

hijkl

m

_ijklm

_jklm

_klm

_lm

_m

0,0 1,0 2,0 T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

g

_bcdef

g

bcdef

gh

cdefg

hi

cdefg

hij

cdefg

hil

cdefg

hij

hijk

cdefg

hijk

cdefg

hijk

cdefg

hijk

defgh

ijk

defgh

ijk

l

efghijk

l

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

TP5

a

_ab

_abc

_bcd

e

_bcd

e

_bcd

e

_bcd

ef

efg

_bcd

efg

bcd

efgh

cde

fghi

cde

fghij

cde

fghil

cde

fghij

cde

fghij

k

cde

fghij

k

cde

fghij

k

cde

fghij

k

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

_fghij

kl

ghijk

lm

hijkl

m

hijkl

m

_ijklm

_jklm

_klm

_lm

_m

0,0 1,0 2,0 T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

_bcdefg

bcdefgh

_cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

m

lm

klm

jklm

ijklm

hijklm

ghijk

lm

fghijk

l

efgh

ijkl

efgh

ijkl

defg

hijk

defg

hijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghij

cdef

ghil

cdef

ghij

cdef

ghi

bcde

fgh

bcde

fg

bcde

fg

bcde

f

bcde

bcd

abc

ab

a

0.0 1.0 2.0

m

lm

klm

jklm

ijkl

m

hijk

lm

hijk

lm

gh

ijkl

m

fgh

ijkl

efg

hijk

l

efg

hijk

l

de

fgh

ijk

de

fgh

ijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hijk

cd

efg

hij

cd

efg

hil

cd

efg

hij

cd

efg

hi

bc

de

fgh

bc

de

fg

bc

de

fg

bc

de

f

bc

de

bc

de

bc

de

bc

d

bc

d

abc

ab

a

0.0 1.0 2.0

m

lm

kl

m

jk

lm

ijk

lm

hi

jk

lm

hi

jk

lm

gh

ijk

lm

fg

hi

jk

l

ef

gh

ijk

l

ef

gh

ijk

l

de

fg

hi

jk

de

fg

hi

jk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ijk

cd

ef

gh

ij

cd

ef

gh

il

cd

ef

gh

ij

cd

ef

gh

i

bc

de

fg

h

bc

de

fg

bc

de

fg

bc

de

f

bc

de

bc

de

bc

de

bc

d

bc

d

abc

ab

a

0.0

1.0

2.0

T P5

a

_ab

_abc

_bcd

_bcde

f

_bcde

fg

_bcde

fg

bcde

fgh

cdef

ghi

cdef

ghij

cdef

ghil

cdef

ghij

ghijk

cdef

ghijk

cdef

ghijk

cdef

ghijk

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

fghijk

l

ghijk

lm

hijklm

ijklm

jklm

klm

lm

m

0,0

1,0 2,0

TP5

a

_ab

_abc

_bcd

e

_bcd

e

_bcd

e

_bcd

ef

_bcd

efg

_bcd

efg

bcd

efgh

cde

fghi

cde

fghi

j

cde

fghi

l

cde

fghi

j

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

cde

fghi

jk

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

_fghi

jkl

ghijk

lm

hijkl

m

hijkl

m

_ijklm

_jklm

_klm

_lm

_m

0,0 1,0 2,0 T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

g

_bcdef

g

bcdef

gh

cdefg

hi

cdefg

hij

cdefg

hil

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hij

hijk

cdefg

hijk

cdefg

hijk

cdefg

hijk

defgh

ijk

defgh

ijk

l

efghijk

l

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0

TP5

a

_ab

_abc

_bcd

e

_bcd

e

_bcd

e

_bcd

ef

efg

_bcd

efg

bcd

efgh

cde

fghi

cde

fghij

cde

fghil

cde

fghij

cde

fghij

k

cde

fghij

k

cde

fghij

k

cde

fghij

k

defg

hijk

defg

hijk

efgh

ijkl

efgh

ijkl

_fghij

kl

ghijk

lm

hijkl

m

hijkl

m

_ijklm

_jklm

_klm

_lm

_m

0,0 1,0 2,0 T P5

a

_ab

_abc

_bcd

_bcde

_bcdef

bcdefg

bcdefgh

cdefghi

cdefghij

cdefghil

cdefghij

cdefghijk

defghijk

efghijkl

fghijkl

ghijklm

hijklm

ijklm

jklm

klm

lm

m

0,0 1,0 2,0