Online Resource 1 The final concentrations of the applied technical enzymes in the matrices wheat
flour, bread and cookies
-Amylase from Xylanase from Lipase from
Thermomyces Thermomyces
Aspergillus
lanuginosus
lanuginosus
oryzae
[ppm]
[ppm]
[ppm]
Flour 0
Flour 1
Flour 2
Flour 3
Flour 4
0.00
2.58
9.03
19.35
38.70
0.00
1.04
3.64
7.80
15.60
0.00
0.36
1.26
2.70
5.40
Bread 0
Bread 1
Bread 2
Bread 3
Bread 4
0.00
1.94
7.61
19.22
38.83
0.00
0.78
3.07
7.75
15.65
0
0.27
1.06
2.68
5.42
Cookie 0
Cookie 1
Cookie 2
Cookie 3
Cookie 4
0.00
2.58
9.03
19.35
38.70
0.00
1.04
3.64
7.80
15.60
0
0.36
1.26
2.7
5.4
Online Resource 2 The recipe for bread making
Component
Rest-Wheat flour
Peanut fat
Sugar
Salt (NaCl)
Yeast
Pre-Mix enzyme
Weight [g]
1381
68
27
41
68
52
Water
Total
889
2500
Online Resource 3 The recipe for cookies
Component
Rest-Wheat flour
Sunflower margarine
Sugar
Baking powder
Pre-Mix enzyme
Water
Total
Weight [g]
255
100
100
9
36
100
600
Online Resource 4 Preparation of calibration standards for HPLC-MS/MS
The peptides were obtained from peptides&elephants GmbH (Potsdam, Germany).
Solvents

Extraction buffer: Dissolve 7.9g Ammonium bicarbonate, 240 g Urea, 0.78 g 1,4-Dithiothreitol in
1000 ml distilled water. All chemicals were from Carl Roth GmbH + Co. KG (Karlsruhe, Germany).

Buffer A: 100ml distilled water + 100ml Acetonitrile (Merck KGaA, Darmstadt, Germany) + 0.2 ml
formic acid (Carl Roth GmbH + Co. KG, Karlsruhe, Germany).

Buffer B: 200 ml distilled water + 0.2 ml formic acid (Carl Roth GmbH + Co. KG, Karlsruhe,
Germany).
Protein standard for digestion control
A stock solution of α-Amylase from Bacillus licheniformis was prepared by dissolving 5.46 mg in 1 ml of
the extraction buffer.
ISTD for α-Amylase from Bacillus licheniformis
A corresponding specific peptide (peptide sequence: EGDSSVANSGLAALITDGPGGAK, MW = 2086.02)
for α-Amylase from Bacillus licheniformis was prepared by dissolving 2.4 mg in 1 ml buffer A to get a
stock solution. 416.7 µl were than diluted to 10 ml with buffer B to give DL1 ISTD solution. This (2 ml
DL1 ISTD) was further diluted with buffer B (8 ml) to produce DL2 ISTD.
Peptide standard for isotope marked peptides
Stock solutions
Peptide
Xylanase 493 heavy
Xylanase 599 heavy
Amylase 329 heavy
Amylase 411 heavy
Lipase 465 heavy
Lipase 575 heavy
Lipase 583 heavy
Peptide
sequence
GWNPGLNAR
TSGTVQTGCH
FDAWAR
ALSSALHER
NWPIYK
SGTLVPVTR
GHDGFTSSWR
ITHTNDIVPR
MW
[g/mol]
995.48
1749.18
Weight
[mg]
2.8
2.3
Volume
buffer A [µl]
500
500
Concentration Stock
solution SL [mg/ml]
5600
4600
994.40
829.90
941.07
1160.52
1177.40
3.9
2.7
2.0
2.0
3.1
500
500
500
500
500
7800
5400
4000
4000
6200
Dilutions
Peptide
Volume SL [µl]
Xylanase 493 heavy
178.6
Xylanase 599 heavy
217.4
Amylase 329 heavy
128.2
Amylase 411 heavy
185.2
Lipase 465 heavy
250.0
Lipase 575 heavy
250.0
Lipase 583 heavy
161.3
Peptide standard for unmarked peptides
DL 1 Isopepmix [100 ppm]
Fill up with buffer B to 10 ml
DL1 – Isopepmix enzymes
Fill up with buffer B to 10 ml
DL1 – Isopepmix lipase
DL 2 Isopepmix [5 ppm]
Fill up 500 µl DL 1 with
buffer B to 10 ml
DL2 – Isopepmix
enzymes
Fill up 500 µl DL 1 with
buffer B to 10 ml
DL2 – Isopepmix lipase
Stock solutions
Peptide
Xylanase 493
Amylase 411
Lipase 465
Lipase 575
Lipase 583
Dilutions
Peptide
sequence
GWNPGLNAR
NWPIYK
SGTLVPVTR
GHDGFTSSWR
ITHTNDIVPR
MW
[g/mol]
985.41
821.37
930.61
1150.33
1167.51
Weight
[mg]
2.5
2.2
2.0
2.2
2.5
Volume
buffer A [µl]
500
500
500
500
500
Concentration Stock
solution SL [mg/ml]
5000
4400
4000
4400
5000
Peptide
Xylanase 493
Amylase 411
Volume SL [µl]
DL 1 Isopepmix [100 ppm]
200.0 Fill up with buffer B to 10 ml
227.3 DL1 – Pepmix enzymes
Lipase 465
Lipase 575
Lipase 583
250.0 Fill up with buffer B to 10 ml
227.3 DL1 – Pepmix lipase
200.0
DL 2 Isopepmix [0.5 ppm]
Fill up 50 µl DL 1 with
buffer B to 10 ml
DL2 – Pepmix enzymes
Fill up 50 µl DL 1 with
buffer B to 10 ml
DL2 – Pepmix lipase
Finally, a further dilution (DL3) was prepared by taking 2 ml DL2 – Pepmix enzymes/lipase and filling up
with buffer B to 10 ml [0.1 ppm]
All the prepared solutions stored in brown bottles at -18°C.
Calibration
Calibration
point (CP)
0
1
2
3
4
5
6
7
Volume DL3 Pepmix
Enzymes/lipase [µl]
2
5
10
20
100
200
400
600
Volume DL2 Isopepmix Volume Volume Final
enzymes/lipase [µl]
DL2 ISTD buffer
concentration
[µl]
B [µl]
for CP [ppm]
40
100
858
0.0002
40
100
855
0.0005
40
100
850
0.0010
40
100
840
0.0020
40
100
760
0.0100
40
100
660
0.0200
40
100
460
0.0400
40
100
260
0.0600
Final concentration for the isotope marked peptides is 0.2 ppm.
Final concentration of the specific peptide (EGDSSVANSGLAALITDGPGGAK) for α-Amylase from Bacillus
licheniformis is 2.0 ppm
Online Resource 5A SDS-PAGE of Lipase FE-01 – the marked protein band was excised, treated and
digested by trypsin and analysed by MALDI-TOF-MS as described in methods section.
Online Resource 5B Peptide calibration standard II for MALDI-TOF-MS
Peptide
Bradykinin 1-7
Angiotensin II
Angiotensin I
Substance P
Bombesin
Renin Substrate
ACTH clip 1-17
ACTH clip 18-39
Somatostatin 28
[M+H]+ Mono isotopic
757.3992
1046.5418
1296.6848
1347.7354
1619.8223
1758.9326
2093.0862
2465.1983
3147.4710
[M+H]+ Average
757.86
1047.19
1297.49
1348.64
1620.86
1760.03
2094.43
2466.68
3149.57
Online Resource 6 Peptides identified (black) in sequence of lipase of Thermomyces lanuginosus by
MALDI-TOF-MS
Sample 1
Sample 2
Sample 3
Online Resource 7 Results of BLAST and Align analysis for the sequence of lipase of Thermomyces
lanuginosus (LIP_THELA) as determined by MALDI-TOF-MS analysis
The Basic Local Alignment Search Tool (BLAST) was applied to find regions of local similarity between
sequences, which can be used to infer functional and evolutionary relationships between sequences as
well as help identify members of gene families. The tool is available online at
http://www.uniprot.org/blast/ and the data was re-checked in March 2015 (original report is available).
The selection data is summarized and limited to the first few hits.
Since the producer of the tested Lipase FE-0 gave the originating organism as from Aspergillus oryzae, an
alignment was conducted of the sequence of Lipase from Thermomyces lanuginosus (LIP_THELA) with
the corresponding hits from Aspergillus oryzae (G9M5R3_ASPNG and I7ZZJ6_ASPO3) with higher
identity similarity (ca. 51 %). The sequence alignment was reproduced using the data in FASTA form
available on analysis with the 'Align' tool available at http://www.uniprot.org/align/. The reproduction
was achieved using the software Jalview (The Barton Group, University of Dundee, Scotland, UK), Vers
2.8. The peptides used for quantification/qualification with HPLC MS/MS analysis are bordered in red.
Code for identity:
Online Resource 8 Sequence alignment of amylases from three relevant sources ‐ Aspergillus
shirousami (AMY_ASPSH), Triticum aestivum (AMY3_WHEAT) and Bacillus licheniformis (AMY_BACLI)
Alignment was conducted online at http://www.uniprot.org/ and the FASTA files were evaluated using
the software Jalview Vers.: 2.8 (The Barton group, University of Dundee, Scotland, UK). The peptides
marked by the red/green frame were those applied for identification purposes.
Code for identity:
Online Resource 9 Sequence alignment of xylanases from the two relevant sources ‐ Thermomyces
lanuginosus (XYNA_THELA) and Triticum aestivum (AMY3_WHEAT)
Alignment was conducted online at http://www.uniprot.org/ and the FASTA files were evaluated using
the software Jalview Vers.: 2.8 (The Barton group, University of Dundee, Scotland, UK). The peptides
marked by the red frame were those applied for identification purposes.
Code for identity:
Online Resource 10 Identified peptides of -amylase in test material bread sample 3 (19 ppm
-amylase from Aspergillus sp) and that of the internal standard peptide (ISTD 496) of the amylase
from Bacillus licheniformis (AMY_BACLI).
Online Resource 11 HPLC-MS/MS analysis for peptides of -amylase in test material bread blank – the
results show the absence of the corresponding identified peptides of -amylase from Aspergillus sp).
The internal standard peptide (ISTD 496) of the amylase from Bacillus licheniformis (AMY_BACLI) is
also shown in the last chromatogram.
Online Resource 12 Calculation of the recovery based on the internal standard peptide (ISTD 496) of
the amylase from Bacillus licheniformis (AMY_BACLI) – example shows illustrates the recovery
determination for lipase.
The concentration of the lipase 465 found in the digest obtained from wheat flour and baked goods can
be determined from the above calibration. The value thus obtained is multiplied by an individual
protein-peptide factor, which represents the mass ratio of the enzyme lipase to the lipase 465 (34.18).
Since the amount administered in the wheat flour or baked goods is known (supporting information
figure S1), the recovery can be calculated in [%]. The protein-peptide factors for -amylase
(corresponding peptide -amylase 411) was 66.72 and for xylanase (corresponding peptide xylanase
493) 24.76 respectively.
Online Resource 13 Calculation of the recovery based on the isotope marked internal peptide
standards for the individual enzymes – example shows illustrates the recovery determination for
-amylase in wheat flour, dough, bread and cookies (overnight digestion).
Calibration curve: Area ratio = concentration of the amylase 411 / concentration of the isotope labelled
amylase 411
Recovery was calculated similar to the method applied in supporting information figure S12.
Online Resource 14 Calculation of the recovery based on the isotope marked internal peptide
standards for the individual enzymes – example shows illustrates the recovery determination for
xylanase in wheat flour, dough, bread and cookies (overnight digestion).
Calibration curve: Area ratio = concentration of the xylanase 493 / concentration of the isotope labelled
xylanase 493
Recovery was calculated similar to the method applied in supporting information figure S12.
Online Resource 15 Calculation of the recovery based on the isotope marked internal peptide
standards for the individual enzymes – example shows illustrates the recovery determination for
lipase in wheat bread and cookies (overnight digestion).
Calibration curve: Area ratio = concentration of the lipase 465 / concentration of the isotope labelled
lipase 465
Recovery was calculated similar to the method applied in supporting information figure S12. White
columns < LOD = level of detection; grey columns < LOQ = Level of quantification.
Online Resource 16 Changes in bread quality by using moderate amount of technical enzymes. The
figures shows bread 0 – bread 4 (left to right) with different amounts of the enzymes as given in the
supporting information figure S1.