Functional Metabolome Analysis of Penicillium roqueforti by Means of

ACS2GO © 2019. ← → → ←. loading. To add this web app to the home screen open the browser option menu and tap on Add to homescreen...
0 downloads 0 Views 2MB Size
Subscriber access provided by UNIV AUTONOMA DE COAHUILA UADEC

Bioactive Constituents, Metabolites, and Functions

Functional Metabolome Analysis of Penicillium roqueforti by Means of DOLC-NMR Richard Hammerl, Oliver Frank, Tina Schmittnägel, Matthias Ehrmann, and Thomas Hofmann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00388 • Publication Date (Web): 05 Apr 2019 Downloaded from http://pubs.acs.org on April 5, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 46

Journal of Agricultural and Food Chemistry

1

Functional Metabolome Analysis of Penicillium

2

roqueforti by Means of DOLC-NMR

3 4 5

Richard Hammerl1, Oliver Frank1, Tina Schmittnägel1, Matthias A. Ehrmann2,

6

Thomas Hofmann13*

7 8 9 1Chair

10

of Food Chemistry and Molecular Sensory Science, Technische Universität

München, Lise-Meitner-Str. 34, D-85354 Freising-Weihenstephan, Germany

11

2Chair

12

of Technical Microbiology, Technische Universität München,

Gregor-Mendel-Str.4, D-85354 Freising-Weihenstephan, Germany, and

13

3Leibniz-Institute

14

for Food Systems Biology at the Technical University of Munich,

Lise-Meitner-Str. 34, D-85354 Freising-Weihenstephan, Germany

15 16 17 18 19

*

20

PHONE

+49-8161-712902

21

FAX

+49-8161-712949

22

E-MAIL

To whom correspondence should be addressed

[email protected]

23 24 25

1 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

26

Page 2 of 46

ABSTRACT

27 28

UPLC-TOF-MS profiling, followed by the recently reported DOLC-NMR and

29

quantitative 1H-NMR spectroscopy led to the differential qualitative analysis and

30

accurate quantitation of L-tryptophan-induced metabolome alterations of Penicillium

31

roqueforti, which is typically used in blue-mould cheese making. Among the 24

32

metabolites identified, two tetrapeptides, namely

33

D-Phe-L-Val-D-Val-L-Phe,

34

first time as metabolites of P. roqueforti. Antimicrobial activity tests showed strong

35

effects of the catabolic L-tryptophan metabolites 3-hydroxyanthranilic acid, anthranilic

36

acid, and 3-indolacetic acid against S. cerevisiae with IC50 values between 15.6 and

37

24.0 µg/mL, while roquefortine C and cis-bis-(methylthio)-silvatin inhibited the growth

38

of Gram negative Escherichia coli and Gram positive Bacillus subtilis with IC50 values

39

between 30.0 and 62.5 µg/mL.

D-Phe-L-Val-D-Val-L-Tyr

and

as well as cis-bis-(methylthio)-silvatin are reported for the

40 41

Keywords: Penicillium roqueforti, P. roqueforti, DOLC-NMR, differential off-line

42

LC-NMR, qHNMR, ERETIC 2, NMR bucket table

43

2 ACS Paragon Plus Environment

Page 3 of 46

45

Journal of Agricultural and Food Chemistry

INTRODUCTION

46 47

The ascomycete P. roqueforti, an aerobe fungus related to the family of

48

Trichocomaceae, is widely used for industrial applications, e.g. the production of

49

enzymes, as well as for the manufacturing of blue mould cheeses, where it

50

contributes to the unique flavor profile of the cheese by the biogeneration of odor-

51

active methyl ketones like 2-pentanone and 2-hexanone,1–3 as well as kokumi

52

enhancing γ-glutamyl dipeptides like γ-Glu-Glu and γ-Glu-Met, respectively.4

53

Studies on the optimal fermentation temperature show the highest metabolic

54

rates for P. roqueforti between 21 and 24 °C and microbial growth of the fungi can be

55

observed up to 32 °C.5,6 Penicillium species have been reported to metabolize amino

56

acids mainly via transamination to give the corresponding α-keto acids, some of which

57

are used for energy supply or a carbon source for more complex secondary

58

metabolites, or via a lyase-catalyzed elimination reaction that, for example, may lead

59

to the release of phenol from the progenitor amino acid L-tyrosine.7,8 In addition,

60

amino acids used as a carbon source when P. roqueforti is incubated in the presence

61

of carbohydrates like

62

fermentations seems to slow-down the general metabolism, e.g. the metabolic

63

generation of odor-active methyl ketones from triacylglycerides has been shown to

64

be significantly favoured when P. roqueforti is fermented in the presence of D-glucose

65

and amino acids when compared to fermentation with D-glucose alone.9,11

D-glucose.9,10

Lacking amino acids in P. roqueforti

66

The spectrum of secondary metabolites generated by P. roqueforti comprises

67

various classes, such as, e.g. alkaloids like roquefortines A-E, marcfortine A-C,

68

agroclacvine,

69

sesquiterpenes like eremofortin A-E, Penicillium roqueforti toxins (PR-toxins), (+)-

isofumigaclavine A

and

B

and

3 ACS Paragon Plus Environment

festuclavine,

respectively,

Journal of Agricultural and Food Chemistry

Page 4 of 46

70

aristolochene, and valencene, and tetracyclic triterpenes like the andrastins A-D next

71

to a range of smaller metabolites like mycophenolic acid, bortyodiplodin and penicillic

72

acid.12–16 Some secondary molecules are used for chemical communication between

73

the cells of the fungus and, at the same time, inhibit microbial growth and affect

74

viability of competing microorganisms and pathogens.12,17 Other metabolites like

75

andrastin A

76

farnesyl-transferase in the RAS protein playing a key role in the control of cell

77

proliferation.12,18–20 Roquefortine C has been demonstrated to have a neurotoxic

78

effect in mice,21 is able to inhibit the growth of Gram-positive bacteria,22 and shows

79

an inhibition of the Cytochrom P450.23 In addition isofumigaclavine A, another

80

representative of a tryptophan derived secondary metabolite16, also showed lethal

81

effects in mice (LD50: 340 mg/kg) due to neurotoxic effects.24 Also mycophenolic acid

82

is well known to show antibacterial activity, but also to exhibit immunosuppressive

83

properties to animals.25,26

84

The biosynthesis of various metabolites like eremofortine A-C or Andrastin A-D starts

85

with acetyl-CoA which is transferred into farnesyl diphosphate (FPP) followed by

86

several enzymatic steps forming the target compound from aristolochene as

87

important intermediat.12,15 All known diketopiperazine derivatives are formed in the

88

beginning via the condensation of two amino acids, like L-tryptophan and L-histidine,

89

providing the cyclic metabolite.27

are

reported

to

exhibit

anticancer

activity

by

inhibiting

the

90

While various studies focused on the toxic secondary metabolites and

91

mycotoxin production of various Penicillium species,28,29 studies on Penicillium (P.)

92

roqueforti, used for the manufacturing of blue mould cheese, have been focused

93

primarily on the formation of secondary metabolites in cheese18 and silage13 or on the

94

volatile metabolome.12,30

4 ACS Paragon Plus Environment

Page 5 of 46

Journal of Agricultural and Food Chemistry

95

To investigate the impact of individual amino acids on the generation of

96

secondary metabolites from P. roqueforti, the aim of the present study was to apply

97

the Differential Off-Line LC-NMR approach (DOLC-NMR), a recently developed off-

98

line coupling of HPLC separation and 1H-NMR spectroscopy supported by automated

99

comparative bucket analyses and quantitative

1H-NMR

to record metabolome

100

changes in Saccharomyces cerevisiae upon nutrient interventions, to capture and

101

quantify amino acid induced metabolome alterations in P. roqueforti. NMR

102

spectroscopy, the method of choice, offers the possibility to detect new and unknown

103

metabolites independent of their ionization behaviour. In combination with a very

104

quick direct metabolite quantitation without using many structural complex internal

105

standards, this method constitutes a very strong tool for metabolome analysis.

106 107

MATERIALS AND METHODS

108 109

Chemicals. The following chemicals were obtained commercially: D-glucose,

110

deuterium oxide, methanol-d4, sodium azide, anthranilic acid, 3-hydroxyanthranilic

111

acid, 3-indolacetic acid, N-formylanthranilic acid were obtained from Sigma-Aldrich

112

(Steinheim, Germany), amino acids, malt extract, potassium hydroxide, potassium

113

dihydrogen phosphate, formic acid, methanol, 2-propanol, acetonitrile were from

114

Merck (Darmstadt, Germany). Water used for fermentation and chromatographic

115

separations was purified with a Milli-Q Integral 5 system (Millipore S.A.S., Molsheim,

116

France). TMSP-d4 was supplied by Euriso-Top (Gif-sur-Yvette, France). Synthetic air

117

was from Westfalen (Westfalen AG, Münster, Germany). Tetrapeptides were

118

obtained from Peptides&Elefants (Henningsdorf, Germany).

119

5 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

120

Penicillium Roqueforti Fermentation. P. roqueforti ATCC10110 (CBS

121

221.30), originally isolated from a blue cheese in USA31, was used in this study. P.

122

roqueforti was obtained in ampoules containing a pellet of the lyophilized fungus as

123

spores. For the cultivation of the active fungi cells, the ampoule was opened and the

124

suspended spores were transferred into a growth medium containing malt extract

125

(4 g) and D-glucose (2 g) in water (100 mL) adjusted to pH 5.5 with 0.1% aqueous

126

NaOH. The mixture was moved on a shaking plate (100 rpm) in a glass flask (1 L)

127

sealed with an air permeable cap for 24 h at room temperature.

128

Fermentation of Penicillium Roqueforti in the Presence of Individual Amino

129

Acids. Aliquots (5 mL) of the solution with germinated spores were spiked with

130

individual proteinogenic

131

(100 mL) and D-glucose (0.15 g, 0.83 mmol), were moved in the flasks (1 L) sealed

132

with an air permeable cap on a shaking plate (100 rpm) for 96 h at 23 °C. In addition,

133

a control experiment was performed without any amino acid. Aliquots (5 µL) of the 21

134

samples were used for UPLC-ESI-TOF/MS screening, performed in five replicates for

135

each sample.

136

L-amino

Fermentation with/without

acids (5 mmol/L) and, after addition of water

L-Tryptophan

(Trp1/Trp0). A solution with the

137

geminated spores and the mycelium of P. roqueforti (as detailed above) was placed

138

in the fermenter (Biostat A Plus fermenter, Sartorius, Göttingen, Germany),

139

containing a solution of L-tryptophan (2 g; 5 mmol/L) and D-glucose (3 g, 16.7 mmol)

140

in 2 L water (Trp1 experiment). In addition, control experiments (Trp0) were performed

141

under the identical conditions without the presence of L-tryptophan. The fermentation

142

was performed under aerobic conditions (synthetic air, 1.3 L/min) whilst stirring

143

(150 rpm) for 96 h at 23 °C. The supernatants were then separated from fungi cells

144

by filtration (0.45 µm, Sartorius Stedium Biotech GmbH; Göttingen, Germany),

6 ACS Paragon Plus Environment

Page 6 of 46

Page 7 of 46

Journal of Agricultural and Food Chemistry

145

freeze-dried, and the residue obtained was taken up in water (15 mL). To capture the

146

differences in the metabolomes of fermentation batches Trp1 and Trp0, the obtained

147

extracts were used for RP18-MPLC separation to collect a total of 39 fractions each

148

in 1 min intervals, which were concentrated in vacuum by means of a HT-12

149

evaporation system (Genevac Limited Ipswich, United Kingdom), the corresponding

150

fractions collected from Trp1 and Trp0 were dissolved in deuterated solvents and then

151

analyzed by means of DOLC-NMR as detailed recently.32

152

Medium Pressure Liquid Chromatography (MPLC). The separation of the

153

fermentation broths (Trp0, Trp1) were performed on a Spot Prep II System (Gilson,

154

Limburg, Germany) equipped with a preparative 250 × 21.2 mm, 5 µm Luna

155

PhenylHexyl column (Phenomenex, Aschaffenburg, Germany) as the stationary

156

phase. The effluent (21.0 mL/min) was monitored at 230 nm. The gradient separation

157

was performed with aqueous formic acid (solvent A, 0.1% formic acid in water,

158

pH = 2.5) and acetonitrile (solvent B) as follows: After isocratic elution for 6 min at

159

2% B, the content of acetonitrile was increased to 15% B within 8 min, then an

160

isocratic step with 15% B for 6 min was performed, then B was increased to 30% B

161

within 12 min and, finally, to 100% B within 5 min, then isocratic for 4 min at 100% B.

162

In total 39 fractions (F3-F41) of 1 min intervals were collected and analyzed by means

163

of DOLC-NMR and LC-MS.

164

(2), and cis-bis-(methylthio)-silvatin (3), isolated from fractions F34, F38, and F41,

165

respectively, were identified for the first time as fermentation products of P. roqueforti.

166

D-Phe-L-Val-D-Val-L-Tyr,

D-Phe-L-Val-D-Val-L-Tyr

(1),

D-Phe-L-Val-D-Val-L-Phe

1, Figure 3: LC-MS (ESI+), m/z = 527.29 [M + H]+;

167

LC-MS/MS (DP = 10 V); UPLC-TOF/MSe, m/z = 120 (100), 219 (59), 247 (20), 182

168

(12), 281 (8), 340 (3); UPLC-TOF/MS, m/z = 527.2866 (measured), m/z = 527.2870

169

(calculated for [C28H39N4O6]+); 1H-NMR (500.13 MHz, MeOD-d4, COSY): δ = 7.36 [m,

7 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 46

170

2H, H-C(6,8)], 7.30 [m, 2H, H-C(5,9)], 7.29 [m, 1H, H-C(7)], 6.99 [d, J = 8.5, 2H, H-

171

C(5´´´,9´´´)], 6.67 [d, J = 8.5, 2H, H-C(6´´´,8´´´)], 4.32 [dd, J = 4.8, 6.7, 1H, H-C(2´´´)],

172

4.23 [d, J =6.3, 1H, H-C(2´´)], 4.08 [d, J =9.3, 1H, H-C(2´)], 3.74 [dd, J =7.6, 1H, H-

173

C(2)], 3.11 [dd, J = 7.5, 13.5, 1H, H-C(3α)], 3.10 [dd, J = 6.8, 13.9, 1H, H-C(3´´´α)],

174

3.01 [dd, J = 7.5, 13.5, 1H, H-C(3β)], 2.84 [dd, J = 4.7, 13.9, 1H, H-C(3´´´β)], 2.22 [m,

175

1H, H-C(3´´)], 1.90 [m, 1H, H-C(3´)], 0.86 [d, J = 6.7, 3H, H-C(4´)], 0.85 [d, J = 6.7,

176

6H, H-C(4´´,5´´)], 0.65 [d, J = 6.7, 3H, H-C(5´)];

177

HSQC, HMBC): δ = 177.6 [C-1´´´], 173.4 [C-1´], 171.9 [C-1´´], 169.7 [C-1], 156.8 [C-

178

7´´´], 135.9 [C-4], 131.8 [C-5´´´,9´´´], 130.6 [C-5,9], 130.1 [C-6,8], 129.5 [C-4´´´], 128.8

179

[C-7], 116.0 [C-6´´´,8´´´], 60.7 [C-2´], 59.7 [C-2´´], 57.3 [C-2´´´], 55.7 [C-2], 38.8 [C-3],

180

37.9 [C-3´´´], 30.6 [C-3´], 29.8 [C-3´´], 20.0 [C-4´], 19.6 [C-4´´], 18.9 [C-5´], 17.9 [C-

181

5´´].

182

D-Phe-L-Val-D-Val-L-Phe,

13C-NMR

(125 MHz, MeOD-d4,

2, Figure 3: LC-MS (ESI+), m/z = 511.30 [M + H]+;

183

LC-MS/MS (DP = 10 V); UPLC-TOF/MSe, m/z = 120 (100), 219 (40), 247 (11), 166

184

(6), 265 (6), 386 (3), 346 (2), 340 (1); UPLC-TOF/MS, m/z = 511.2918 (measured),

185

m/z = 511.2920 (calculated for [C28H39N4O5]+); 1H-NMR (500.13 MHz, MeOD-d4,

186

COSY): δ = 7.36 [m, 2H, H-C(6,8)], 7.30 [m, 1H, H-C(7)], 7.28 [m, 2H, H-C(5,9)], 7.22

187

[m, 2H, H-C(6´´´,8´´´)], 7.18 [m, 1H, H-C(7´´´)], 7.17 [m, 2H, H-C(5´´´,9´´´)], 4.42 [dd,

188

J = 4.8, 7.2, 1H, H-C(2´´´)], 4.20 [d, J =6.5, 1H, H-C(2´´)], 4.07 [d, J =9.4, 1H, H-C(2´)],

189

3.75 [dd, J =7.6, 1H, H-C(2)], 3.21 [dd, J = 4.8, 13.8, 1H, H-C(3´´´α)], 3.10 [dd, J =

190

7.6, 13.6, 1H, H-C(3α)], , 3.00 [dd, J = 7.6, 13.6, 1H, H-C(3β)], 2.90 [dd, J = 7.2, 13.8,

191

1H, H-C(3´´´β)], 2.19 [m, 1H, H-C(3´´)], 1.90 [m, 1H, H-C(3´)], 0.84 [d, J = 6.7, 3H, H-

192

C(4´)], 0.83 [d, J = 6.7, 6H, H-C(4´´,5´´)], 0.69 [d, J = 6.7, 3H, H-C(5´)];

193

(125 MHz, MeOD-d4, HSQC, HMBC): δ = 177.3 [C-1´´´], 173.2 [C-1´], 171.6 [C-1´´],

194

169.8 [C-1], 139.3 [C-4´´´], 135.7 [C-4], 130.5 [C-5´´´,9´´´], 130.3 [C-5,9], 129.9 [C-

8 ACS Paragon Plus Environment

13C-NMR

Page 9 of 46

Journal of Agricultural and Food Chemistry

195

6,8], 129.0 [C-6´´´,8´´´], 128.8 [C-7], 127.2 [C-4´´´], 60.6 [C-2´], 59.6 [C-2´´], 56.9 [C-

196

2´´´], 55.6 [C-2], 37.5 [C-3´´´], 37.1 [C-3], 30.5 [C-3´], 29.6 [C-3´´], 19.9 [C-4´´], 19.6

197

[C-4´], 19.2 [C-5´], 17.7 [C-5´´].

198

Cis-bis-(methylthio)-silvatin, 3, Figure 3: LC-MS (ESI+), m/z = 431.15

199

[M + Na]+; LC-MS/MS (DP = 10 V); UPLC-TOF/MSe, m/z = 337 (100), 107 (58), 217

200

(40), 245 (15), 293 (4), 361 (3); UPLC-TOF/MS, m/z = 431.1453 (measured), m/z =

201

431.1439 (calculated for [C20H28N2O3S2Na]+);

202

COSY): δ = 6.99 [d, J = 8.7, 2H, H-C(9/13)], 6.80 [d, J = 8.7, 2H, H-C(10/12)], 5.42

203

[ddqq, J = 1.3, 6.5, 1H, H-C(15)], 4.48 [d, J = 6.5, 2H, H-C(14)], 4.46 [s, 1H, H-C(3)],

204

3.34 [d, J = 14.1, 1H, H-C(7α)], 3.22 [s, 3H, H-C(19)], 3.20 [d, J = 14.1, 1H, H-C(7β)],

205

3.00 [s, 3H, H-C(20)], 2.29 [s, 3H, H-C(20)], 2.14 [s, 3H, H-C(22)], 1.78 [s, 3H, H-

206

C(18)], 1.73 [s, 3H, H-C(17)];

207

166.9 [C-2], 166.3 [C-5], 160.1 [C-11], 138.8 [C-16], 131.8 [C-9/13], 127.4 [C-8], 121.3

208

[C-15], 115.9 [C-10/12], 76.7 [C-6], 66.4 [C-3], 66.3 [C-14], 42.9 [C-7], 33.9 [C-20],

209

30.9 [C-19], 25.9 [C-18], 18.2 [C-17], 16.4 [C-21], 13.6 [C-22].

210

13C-NMR

1H-NMR

(500.13 MHz, MeOD-d4,

(125 MHz, MeOD-d4, HSQC, HMBC): δ =

Ultra Performance Liquid Chromatography Electrospray Ionization-

211

Time-of-Flight

Mass

Spectrometry

(UPLC-ESI-TOF/MS).

212

supernatant (5 µL) obtained from the P. roqueforti fermentation broths containing

213

individual amino acids were analysed by UPLC-ESI-TOF/MS on a Waters Synapt

214

G2-S HDMS mass spectrometer (Waters, Manchester, United Kingdom) coupled to

215

an Acquity UPLC core system (Waters, Milford, MA, USA) equipped with a 2 x

216

150 mm, 1.7 μm, BEH C18 column (Waters, Manchester, United Kingdom).

217

Chromatography was performed with a flow rate of 0.4 mL/min at 50 °C using a

218

solvent gradient starting with 99% aqueous formic acid (0.1% in water, pH = 2.5;

219

solvent A) and 1% acetonitrile containing 0.1% formic acid (solvent B) and increasing

9 ACS Paragon Plus Environment

Aliquots

of

the

Journal of Agricultural and Food Chemistry

220

solvent B to 100% within 4.5 min. Scan time for the MSe method (centroid) was set

221

to 0.1 s. Analysis were performed with negative and positive ESI+ in high-resolution

222

mode using the ion source parameters given in parenthesis: capillary voltage

223

(−2.0 kV), sampling cone (50 V), source off set (30 V), source temperature (120 °C),

224

desolvation temperature (450 °C), cone gas flow (2 L/h), nebulizer gas (6.5 bar),

225

desolvation gas (800 L/h). Data processing was performed by using MassLynx 4.1

226

SCN 9.16 (Waters, Manchester, United Kingdom) and the elemental composition tool

227

for determining the accurate mass. All data were lock mass corrected on the

228

pentapeptide leucine enkephaline (m/z = 554.2615, [M – H]-; m/z = 556.2771,

229

[M + H]+) in a solution (1 ng/μL) of acetonitrile/0.1% formic acid in water (1/1, v/v).

230

Scan time for the lock mass was set to 0.3 s, at intervals of 15 and 3 scans to average

231

with a mass window of ± 0.3 Da. Calibration of the Synapt G2-S in the range from

232

m/z 50 to 1200 was performed using a solution of sodium format (5 mmol/L) in

233

2-propanol/water (9:1, v/v). The collision energy ramp for MSe was set from 20 to

234

40 eV. The raw data of all fungi samples and replicates obtained from

235

UPLC-ESI-TOF/MS analysis were processed with Progenesis QI using the following

236

workflow: data import, review alignment, experiment design setup, peak picking (all

237

runs, limits automatic, sensitivity 3), review deconvolution, review compounds,

238

compound statistics and retention time limits 0.5-4.75 min. Compounds used for

239

principal component analysis (PCA) were filtered by means of ANOVA p value ≤ 0.05

240

and a fold change of ≥ 2. The processed data were exported to EZinfo, where the

241

matrix was analysed by PCA with pareto scaling.33,34 The same UPLC-TOF/MS

242

system with an identical solvent gradient was used for the determination of the exact

243

masses from the metabolites in the collected fractions.

10 ACS Paragon Plus Environment

Page 10 of 46

Page 11 of 46

Journal of Agricultural and Food Chemistry

244

Nuclear Magnetic Resonance (NMR) Spectroscopy. 1D- and 2D-NMR

245

(COSY, HSQC, HMBC) experiments of the supernatant fractions of the fermentation

246

broth and purified compounds, respectively, were recorded at 300 K on a Bruker

247

AVANCE III 500 MHz System equipped with a cryo-TCI Probe and Topspin 3.2

248

software.35

249

DOLC-NMR Analysis. After concentration of each MPLC fraction in vacuum by

250

means of a HT-12 evaporation system (Genevac Limited, Ipswich, UK), the residues

251

obtained from fractions F3 - F23 were dissolved in D2O (1 mL) and aliquots (540 µL)

252

were, then, prior to NMR analysis, mixed with an aliquot (60 µL) of NMR-buffer, which

253

was prepared by dissolving KH2PO4 (10.2 g) in D2O (40 mL), adding KOH (1.5 g),

254

TMSP-d4 (50 mg), and NaN3 (5 mg), followed by pH-adjustment 7.0 with a solution of

255

KOH (4 mol/L) in D2O and making up to 50 mL with D2O. The more hydrophobic

256

fractions F24 - F41 were dissolved in MeOD-d4 (1 mL) and aliquots (600 µL) used for

257

NMR analysis. The individual fractions were analysed in 5 mm x 7’’ NMR tubes

258

(Z107374 USC tubes, Bruker, Faellanden, Switzerland). A 1H-NMR spectrum was

259

acquired using the Bruker standard water suppression pulse sequence (1D

260

noesygppr1d).36 The 90° pulse length (P1), power level for presaturation of the water

261

resonance (PL9) and O1 were adjusted individually on each sample and 16 scans

262

(NS) with 4 dummy scans (DS) were collected into 64 K data points using a spectral

263

width of 10273.97 Hz. The relaxation time (T1) was set to 20 s to allow all excited

264

nuclei to re-establish their equilibrium z-magnetization prior to the application of the

265

next pulse.35 To ensure high-quality spectra, the NMR probe was manually tuned and

266

matched (atmm) to 50 Ω resistive impedance to minimize radio frequency (RF)

267

reflection, with the sample in place. After automatic optimization of the lock phase,

268

each sample was shimmed (z1 − z5, xyz, z1 − z5), the 90° pulse width was determined

11 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

269

individually for each sample using the AU program pulsecal sn. All spectra were

270

acquired without spinning the sample and referenced to TMSP-d4 (0.0 ppm, fraction

271

F3-F23) or MeOD-d4 (3.31 ppm; fraction F24-F41). The free induction decay (FID)

272

was multiplied with a 0.3 Hz exponential line-broadening factor and zero-filled prior

273

to Fourier transformation. In case that the result of the automatic phase correction

274

with apk0.noe was not satisfying, a careful manual zero- and first-order phase

275

correction was performed. Baseline correction was done automatically with the

276

command absn. Integration was carried out manually and whenever required;

277

adjustment of the integrals was executed by the software functions SLOPE and

278

BIAS.35

279

NMR Bucketing. Corresponding to literature,37,38 NMR-buckets were calculated

280

using the Amix Viewer V3.9.13 Software (Bruker, Rheinstetten, Germany). Each

281

spectrum was referenced to TMSP-d4 (0.0 ppm; fraction F3-F23) or MeOD-d4

282

(3.31 ppm; fraction F24-F41). After checking the baseline offset and application of the

283

underground removal tool, the spectra were used to determine the buckets. Covering

284

the chemical shift region from -1 to 11 ppm, the width of each bucket was set to

285

0.1 ppm and the area between 4.5 and 5 ppm was excluded from bucketing (water

286

signal). The calculation of the absolute integral value for each of the 115 buckets was

287

performed successfully when the signal to noise ratio was higher than 10. The noise

288

was calculated in the region from 10 to 11 ppm, where no signals appeared. The

289

corresponding buckets from the fermenations Trp1 and Trp0, showing an integral ratio

290

(Trp1/Trp0) of >2 or 98% LC-MS) and analyzed by means of a

544

serial dilution microplate method39 to determine the minimum inhibitory concentration

545

(MIC) and the half-maximal inhibitory concentration (IC50) for each compound when

546

applied to Gram negative E. coli, the Gram positive B. subtilis, and the yeast

547

S. cerevisiae as test organisms.

548

Roquefortine C showed a MIC value of 62.5 and a IC50 value of 31.5 µg/mL for

549

B. subtilis (ATCC 6633), respectively (Table 2), thus being well in line with data

550

reported in literature (0.1 mg/mL) for Gram positive B. subtilis (IMM 313).54 For E. coli

551

(ATCC 23226) a IC50 value of 62.5 µg/mL and a MIC value of 125 µg/mL could be

552

detected. Previously performed studies on E. coli regarding an inhibitory effect

553

caused by roquefortine C showed no effect against E. coli (ATCC 11775) at

554

100 µg/mL.22 Another undefined E.coli was not inhibited at 30 µg/disk.55 Comparing

555

the strains to each other, differences between the microorganisms can be observed

556

regarding the occurring enzymes which may explain the differences in the growth

557

rates in the presence of the inhibitor.

558

In comparison, cis-bis-(methylthio)-silvatin (3) showed a lower IC50 value of 30 µg/mL

559

for for E. coli and B. subtilis, respectively. Interestingly, the catabolic L-tryptophan

560

metabolites anthranilic acid (4), 3-indolacetic acid (5) and 3-hydroxyanthranilic acid

561

(6), which induced growth inhibition of the yeast S. cerevisiae with a MIC value of

562

62.5, 31.5 and 31.5 µg/mL, respectively, while N-formylanthranilic acid (11) showed

563

a significant higher MIC value at 250 µg/mL. These data confirm previous reports on

564

the inhibitory activity of 3-hydroxyanthranilic acid on the growth of S. aureus,

565

S. epidermidis, respectively.56 An inhibitory effect on the fungi C. albicans was not

23 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 24 of 46

566

detectable56,57 but the inhibition of S. cerevisiae has been reported for the first time.

567

The mode of action is probably similar to preservatives due to structural similarity.

568

The weak acid enters the microorganism and after the dissociation step, the

569

metabolism is disturbed because of a intracellular reduced pH value.58

570

In this study the growth of E. coli (ATCC 23226) was not affected by

571

3-hydroxyanthranilic acid. Data from literature showed a inhibition of another strain of

572

E. coli (ATCC 11775) at 102.4 µg/mL.56 Due to different enzymatic systems between

573

the analysed strains, the differences may be explained.

574

Moreover, anthranilic acid is reported as effective preservative for animal fodder in

575

concentration of 0.3 to 0.4%59 and suppresses cell proliferation of mesangial cells by

576

32% at 10-6 M.60 The sesquiterpene eremofortin B (8) showed a MIC value of

577

125 µg/mL for the growth inhibition of S. cerevisiae, which has not yet been reported

578

in literature. Also andrastin A (10) showed inhibited the growth of S. cerevisiae (MIC

579

= 62.5 µg/mL), which may be explained by the inhibition of the protein

580

farnesyl-transferase as reported for eukaryotic cells.20 Comparatively, high IC50

581

values of 187

582

tetrapeptides

583

acids with D-conformation are formed via racemisation of L-amino acids. Peptides

584

containing the L-conformer are more stable against proteolytic degradation, which

585

leads to higher half-life periods in organisms.61 Many antibiotics like penicillin G or

586

food toxins like isocereulides derivatives own D-amino acids.62,63 In addition, small

587

peptides with aromatic residues of D-phenylalanine, D-tyrosine or D-tryptophan are

588

described to have a high binding possibility to proteins accompanied with an inhibition

589

of its functionality, respectively.64

and 250 µg/mL, respectively, were found for the identified

D-Phe-L-Val-D-Val-L-Tyr

(1) and

D-Phe-L-Val-D-Val-L-Phe

24 ACS Paragon Plus Environment

(2). Amino

Page 25 of 46

Journal of Agricultural and Food Chemistry

590

In conclusion, DOLC-NMR spectroscopy, followed by qHNMR using

591

ERETIC 2, has been successfully used to monitor metabolome alterations in

592

P. roqueforti induced upon an intervention with

593

metabolites identified, two tetrapeptides, namely

594

D-Phe-L-Val-D-Val-L-Phe

595

for the first time as metabolites of P. roqueforti. Antimicrobial activity tests showed

596

strong effects of the catabolic

597

3-indolacetic acid (5) and 3-hydroxyanthranilic acid (6) against S. cerevisiae with IC50

598

values between 15.6 and 24.0 µg/mL. In comparison, roquefortine C (7) and

599

cis-bis-(methylthio)-silvatin (3) inhibited the growth of Gram negative E. coli and

600

Gram positive B. subtilis with IC50 values between 30.0 and 62.5 µg/mL. Future

601

studies will focus on the one hand on the concentrations of these compounds in

602

P. roqueforti fermented dairy products like blue mould cheeses and on the other hand

603

on inhibitory effects of the identified compounds regarding pathogens and food

604

related microorganisms.

L-tryptophan.

Among the 24

D-Phe-L-Val-D-Val-L-Tyr

(1) and

(2), as well as cis-bis-(methylthio)-silvatin (3) are reported

L-tryptophan

metabolites anthranilic acid (4),

605 606 607

SUPPORTING INFORMATION AVAILABLE

608

Fermentation conditions and NMR data are available free of charge via the Internet

609

at http://pubs.acs.org.

610 611 612 613

25 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

614

Table 1.

Concentration of Key Metabolites in the P. roqueforti ferment in the absence (control, Trp0) and presence of L-tryptophan (Trp1).

615

conc. (μmol/L) in

metabolite

a

Trp1

Trp0

D-Phe-L-Val-D-Val-L-Tyr

(1)

n.d.a

2.4 (±0.2)

D-Phe-L-Val-D-Val-L-Phe

(2)

n.d.a

0.53 (±0.05)

cis-bis-(methylthio)-silvatin (3)

0.86 (±0.09)

n.d.a

anthranilic acid (4)

29.4(±3.0)

n.d.a

3-indolacetic acid (5)

4.1 (±0.4)

n.d.a

3-hydroxyanthranilic acid (6)

442.7 (±45.6)

n.d.a

roquefortine C (7)

1.6 (±0.2)

0.53 (±0.05)

eremofortin B (8)

n.d.a

1.7 (±0.2)

scytalone (9)

n.d.a

1.6 (±0.2)

andrastin A (10)

0.79 (±0.08)

0.92 (±0.09)

67.4 (±7.0)

n.d.a

trehalose

15.9 (±1.7)

14.4 (±1.5)

nicotinic acid

21.0 (±2.2)

n.d.a

uridine

6.5 (±0.7)

n.d.a

tyrosine

6.5 (±0.7)

1.8 (±0.2)

fumaric acid

3.5 (±0.4)

n.d.a

succinic acid

20.2 (±2.1)

6.4 (±0.7)

β-hydroxyisobutyric acid

3.9 (±0.4)

7.0 (±0.7)

kynurenine

7.9 (±0.8)

n.d.a

isopropylmalic acid

2.4 (±0.2)

n.d.a

catechol

58.1 (±6.0)

n.d.a

tyrosol

n.d.a

0.60 (±0.06)

kynurenic acid

43.1 (±4.4)

n.d.a

p-hydroxy-2-phenylacetic acid

2.4 (±0.2)

n.d.a

N-formylanthranilic

616

Page 26 of 46

acid (11)

not detectable

617

26 ACS Paragon Plus Environment

Page 27 of 46

618

Journal of Agricultural and Food Chemistry

Table 2.

Minimum Inhibitory Concentration (MIC) and Half-Maximal Inhibitory

619

Concentration (IC50) of P. roqueforti Metabolites Isolated from the

620

Fermentation broths Trp0 and Trp1 against different microorganisms. MIC and IC50 values (µg/mL) metabolite S. cerevisiae IC50

MIC a

D-Phe-L-Val-D-Val-L-Tyr

(1)

187

n.i.

D-Phe-L-Val-D-Val-L-Phe

(2)

250

n.i.

b

a b

B. subtilis IC50 a

n.i.

a

E. coli

MIC a

n.i.

a

n.i.

n.i. n.i.

b

IC50 a

n.i.

a

MIC a

n.i.

a

n.i.

n.i.

30

n.i.

250

n.i.

187

250

b

cis-bis-(methylthio)-silvatin (3)

n.i.

n.i.

30

anthranilic acid (4)

24

62.5

n.i.

n.i.

3-indolacetic acid (5)

15.6

31.5

250

n.i.

3-hydroxyanthranilic acid (6)

15.6

31.5

250

n.i.

n.i.

n.i.

roquefortine C (7)

250

n.i.

31.5

62.5

62.5

125

eremofortin B (8)

62.5

125

250

n.i.

250

n.i.

scytalone (9)

n.i.

n.i.

n.i.

n.i.

andrastin A (10)

47

62.5

200

n.i.

N-formylanthranilic

b

a

b

a

b

a

acid (11)

a a a

a b a

a

b

a

a

a b

n.i.

n.i.

250

n.i.

a

a a

621

225 250 n.i. n.i. 250 n.i. a No inhibition observable until a tested maximum concentration of 250 µg/mL; b No

622

inhibition observable until a tested maximum concentration of 60 µg/mL.

623

27 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

624

Page 28 of 46

Figure Captions

625 626

Figure 1. Principle component Analysis of UPLC-ESI-TOF/MS full scan data

627

(50−1200 Da, ESI−) of medium broths of P. roqueforti fermented for 96 h at 23 °C in

628

the absence and presence of individual proteinogenic L-amino acids (five replicates

629

per sample).

630 631

Figure 2. (A) RP-MPLC chromatograms of the P. roqueforti fermentation performed

632

in the presence (Trp1) and absence of L-tryptophan (Trp0); (B) Differential NMR

633

bucket analysis of fractions F3−F41 showing the integral ratio (Trp1/Trp0) and

634

chemical shift of metabolites affected by the L-tryptophan intervention.

635 636

Figure 3.

Chemical

structures

of

identified

secondary

metabolites

637

D-Phe-L-Val-D-Val-L-Tyr

638

(3), anthranilic acid (4), 3-indolacetic acid (5), 3-hydroxyanthranilic acid (6),

639

roquefortine C (7), eremofortin B (8), scytalone (9), andrastin A (10) and

640

N-formylanthranilic

(1), D-Phe-L-Val-D-Val-L-Phe (2), cis-bis-(methylthio)-silvatin

acid (11), respectively.

641 642

Figure 4. 1H-NMR (500.13 MHz, MeOD-d4, 300 K, zg30) excerpts (6.5 - 7.7 ppm, 3.5

643

- 4.5 ppm) of (A) D-Phe-L-Val-D-Val-L-Tyr (1) isolated from Trp0 fermentation fraction

644

F34,

645

D-Phe-L-Val-D-Val-L-Phe

646

the synthetic reference D-Phe-L-Val-D-Val-L-Phe; 1H-NMR signal assignment is done

647

according to Figure 3.

(B)

the

synthetic

reference

D-Phe-L-Val-D-Val-L-Tyr,

(C)

(2) isolated from the Trp0 fermentation fraction F38, and (D)

28 ACS Paragon Plus Environment

Page 29 of 46

Journal of Agricultural and Food Chemistry

648

Figure 5. Excerpt of the HMBC spectrum (500.13 MHz, 125 MHz, MeOD-d4, 300 K;

649

3.7-4.4 ppm, 125-180 ppm) of D-Phe-L-Val-D-Val-L-Tyr (1) showing the sequence of

650

the single amino acids in the tetrapeptide backbone.

651

with arrows.

2/3J

H-C-correlations

are marked

652 653

Figure 6. (A) MSe spectra (ESI+) of D-Phe-L-Val-D-Val-L-Phe (2) including the exact

654

mass, calculated elemental composition and chemical formula with a1/2/3, b1/2/3 and

655

y1/2/3 cleavage of the tetrapeptide; (B) MS2 spectra (ESI+) of peptide 6 showing the

656

cleavage fragments.

657 658

Figure 7. Excerpt of the HMBC spectrum (500.13 MHz, 125 MHz, MeOD-d4, 300 K;

659

2.8-7.2 ppm, 10-180 ppm) of cis-bis-(methylthio)-silvatin (3) showing the key

660

correlations of H-C(3), H-C(7) and H-C(14) marked with arrows.

2/3J

H-C

661 1H-NMR

662

Figure 8. Quantitative

spectroscopy (500.13 MHz, MeOD-d4, 300 K,

663

noesygppr1d) to determine the concentration of andrastin A (10) via the quantifier

664

signal at 5.24 ppm (H-(11), s, 1H) and cis-bis-(methylthio)-silvatin (3) via the quantifier

665

signal at 6.80 ppm (H-(10/12), d, 2H) in fraction F41.

666 667 668

29 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

669

Hammerl et al. (Figure 1)

670 671

672 673 674 675 676 677 678 679

30 ACS Paragon Plus Environment

Page 30 of 46

Page 31 of 46

680

Journal of Agricultural and Food Chemistry

Hammerl et al. (Figure 2)

681 682

683 684 685

31 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

686

Hammerl et al. (Figure 3)

687 688

689 690 691 692 693

32 ACS Paragon Plus Environment

Page 32 of 46

Page 33 of 46

694

Journal of Agricultural and Food Chemistry

Hammerl et al. (Figure 4)

695 696

697 698 699 700 701 702 703 704 705 706

33 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

707

Hammerl et al. (Figure 5)

708

709 710 711 712 713

34 ACS Paragon Plus Environment

Page 34 of 46

Page 35 of 46

714

Journal of Agricultural and Food Chemistry

Hammerl et al. (Figure 6)

715 716

717 718 719 720

35 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

721

Hammerl et al. (Figure 7)

722

723 724 725

36 ACS Paragon Plus Environment

Page 36 of 46

Page 37 of 46

726

Journal of Agricultural and Food Chemistry

Hammerl et al. (Figure 8)

727

728 729 730 731 732 733 734 735 736 737 738

37 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

739

References

740

(1) Dwivedi, B. K.; Kinsella, J. E. Carbonyl production from lipolyzed milk fat by the

741

continuous mycelial of Penicillium roqueforti, J. Food Sci. 1974, 39, pp. 83–87.

742

(2) Kinsella, J. E.; Hwang, D. Biosynthesis of flavors by Penicillium roqueforti,

743

Biotechnol. Bioeng. 1976, 18, pp. 927–938.

744

(3) Qian, M.; Nelson, C.; Bloomer, S. Evaluation of fat-derived aroma compounds in

745

blue cheese by dynamic headspace GC/Olfactometry-MS, J. Amer. Oil Chem. Soc.

746

2002, 79, pp. 663–667.

747

(4) Toelstede, S.; Hofmann, T. Kokumi-active glutamyl peptides in cheeses and their

748

biogeneration by Penicillium roquefortii, J. Agric. Food Chem. 2009, 57, pp. 3738–

749

3748.

750

(5) Cuppers, H. G.; Oomes, S.; Brul, S. A Model for the Combined Effects of

751

Temperature and Salt Concentration on Growth Rate of Food Spoilage Molds, Appl.

752

Environ. Microbiol. 1997, 63, pp. 3764–3769.

753

(6) Li, Y.; Wadso, L.; Larsson, L. Impact of temperature on growth and metabolic

754

efficiency of Penicillium roqueforti--correlations between produced heat, ergosterol

755

content and biomass, J. Appl. Microbiol. 2009, 106, pp. 1494–1501.

756

(7) Yvon, M.; Rijnen, L. Cheese flavour formation by amino acid catabolism, Int. Dairy

757

J. 2001, 11, pp. 185–201.

758

(8) Adour, L.; Aziza, M.; Couriol, C.; Amrane, A. Amino acids as carbon, energy and

759

nitrogen sources for Penicillium camembertii, J. Chem. Technol. Biotechnol. 2006,

760

81, pp. 573–579.

761

(9) Meyers, E.; Knight, S. G. Studies on the Nutrition of Penicillium roqueforti, Appl.

762

Microbiol. 1958, 6, pp. 174–178.

38 ACS Paragon Plus Environment

Page 38 of 46

Page 39 of 46

Journal of Agricultural and Food Chemistry

763

(10) Ayati, F.; Aziza, M.; Maachi, R.; Amrane, A. The Substrate Carbon Consumption

764

and Metabolite Production to Describe the Growth of Geotrichum candidum and

765

Penicillium camemberti on Glucose and Amino Acids, Food Technol. Biotechnol.

766

2010, 48, pp. 79–85.

767

(11) Lawrence, R. C. The Metabolism of Triglycerides by Spores of Penicillium

768

roqueforti, J. gen. Microbiol. 1967, 46, pp. 65–76.

769

(12) Mioso, R.; Toledo Marante, F. J.; Herrera Bravo de Laguna, I Penicillium

770

roqueforti: a multifunctional cell factory of high value-added molecules, J. Appl.

771

Microbiol. 2015, 118, pp. 781–791.

772

(13) Nielsen, K. F.; Sumarah, M. W.; Frisvad, J. C.; Miller, J. D. Production of

773

Metabolites from the Penicillium roqueforti Complex, J. Agric. Food Chem. 2006, 54,

774

pp. 3756–3763.

775

(14) Moreau, S.; Biguet, J. Structure et stereochimie des Sesquieterpenes de

776

Penicillium roqueforti PR Toxine et Eremofortines A, B, C, D, E, Tetrahedron. 1980,

777

36, pp. 2989–2997.

778

(15) Riclea, R.; Dickschat, J. S. Identification of intermediates in the biosynthesis of

779

PR toxin by Penicillium roqueforti, Angew. Chem. 2015, 54, pp. 12167–12170.

780

(16) Fernández-Bodega, Á.; Álvarez-Álvarez, R.; Liras, P.; Martín, J. F. Silencing of

781

a second dimethylallyltryptophan synthase of Penicillium roqueforti reveals a novel

782

clavine alkaloid gene cluster, Appl. Microbiol. Biotechnol. 2017, 101, pp. 6111–6121.

783

(17) Martín, J. F.; Casqueiro, J.; Liras, P. Secretion systems for secondary

784

metabolites: how producer cells send out messages of intercellular communication,

785

Curr. Opin. Microbiol. 2005, 8, pp. 282–293.

39 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

786

(18) Nielsen, K. F.; Dalsgaard, P. W.; Smedsgaard, J.; Larsen, T. O. Andrastins A-D,

787

Penicillium roqueforti Metabolites consistently produced in blue-mold-ripened

788

cheese, J. Agric. Food Chem. 2005, 53, pp. 2908–2913.

789

(19) Uchida, R.; Shiomi, K.; Inokoshi, J.; Sunazuka, T.; Tanaka, H.; Iwai, Y.;

790

Takayanagi, H.; Omura, S. Andrastins A ~ C, New Protein Farnesyltransferase

791

Inhibitors Produced by Penicillium sp. FO-3929II. Structure Elucidation and

792

Biosynthesis, J. Antibiot. 1996, 49, pp. 418–424.

793

(20) Overy, D. P.; Larsen, T. O.; Dalsgaard, P. W.; Frydenvang, K.; Phipps, R.; Munro,

794

M. H. G.; Christophersen, C. Andrastin A and barceloneic acid metabolites, protein

795

farnesyl transferase inhibitors from Penicillium albocoremium: Chemotaxonomic

796

significance and pathological implications, J. Mycopathol. Res. 2005, 109, pp. 1243–

797

1249.

798

(21) Arnold, D. L.; Scott, P. M.; McGuire, P. F.; Harwig, J.; Nera, E. A. Acute toxicity

799

studies on Roquefortine and PR Toxin, metabolites of Penicillium Roqueforti, in the

800

mouse, Food Cosmet. Toxicol. 1978, 16, pp. 369–371.

801

(22) Kopp, B.; Rehm, H. J. Antimicrobial action of roquefortine, Eur. J. Appl. Microbiol.

802

Biotechnol. 1979, 6, pp. 397–401.

803

(23) Aninat, C.; Hayashi, Y.; André, F.; Delaforge, M. Molecular Requirements for

804

Inhibition of Cytochrome P450 Activities by Roquefortine, Chem. Res. Toxicol. 2001,

805

14, pp. 1259–1265.

806

(24) Ohmomo, S.; Sato, T.; Utagawa, T.; Abe, M. Isolation of Festuclavine and three

807

new indole alkaloids, Roquefortine A, B and C from the cultures of Penicillium

808

roqueforti, Agric. Biol. Chem. 1975, 39, pp. 1333–1334.

809

(25) Bentley, R. Mycophenolic Acid. A One Hundred Year Odyssey from Antibiotic to

810

Immunosuppressant, Chem. Rev. 2000, 100, pp. 3801–3826. 40 ACS Paragon Plus Environment

Page 40 of 46

Page 41 of 46

Journal of Agricultural and Food Chemistry

811

(26) Adams, E.; Todd, G.; Gibson, W. Long-Term Toxicity Study of MycophenolicAcid

812

in Rabbits, Toxicol. Appl. Pharmacol. 1975, 34, pp. 509–512.

813

(27) Gu, B.; He, S.; Yan, X.; Zhang, L. Tentative biosynthetic pathways of some

814

microbial diketopiperazines, Appl. Microbiol. Biotechnol. 2013, 97, pp. 8439–8453.

815

(28) Fontaine, K.; Hymery, N.; Lacroix, M. Z.; Puel, S.; Puel, O.; Rigalma, K.; Gaydou,

816

V.; Coton, E.; Mounier, J. Influence of intraspecific variability and abiotic factors on

817

mycotoxin production in Penicillium roqueforti, Int. J. Food Microbiol. 2015, 215,

818

pp. 187–193.

819

(29) Garcia-Estrada, C.; Martin, J.-F. Biosynthetic gene clusters for relevant

820

secondary metabolites produced by Penicillium roqueforti in blue cheeses, Appl.

821

Microbiol. Biotechnol. 2016, 100, pp. 8303–8313.

822

(30) Karlshoj, K.; Larsen, T. O. Differentiation of species from the Penicillium

823

roqueforti group by volatile metabolite profiling, J. Agric. Food Chem. 2005, 53,

824

pp. 708–715.

825

(31) Shetty, A. S.; Gaertner, F. H. Kynureninase-Type enzymes of Penicillium

826

roqueforti, Aspergillus niger, Rhizopus stolonifer, and Pseudomonas fluorescens:

827

Further evidence for distinct Kynureninase and Hydroxykynureninase activities, J.

828

Bacteriol. 1975, 122, pp. 235–244.

829

(32) Hammerl, R.; Frank, O.; Hofmann, T. Differential Off-line LC-NMR (DOLC-NMR)

830

Metabolomics

831

Saccharomyces cerevisiae, J. Agric. Food Chem. 2017, 65, pp. 3230–3241.

832

(33) Stark, T.; Marxen, S.; Rutschle, A.; Lucking, G.; Scherer, S.; Ehling-Schulz, M.;

833

Hofmann, T. Mass spectrometric profiling of Bacillus cereus strains and quantitation

834

of the emetic toxin cereulide by means of stable isotope dilution analysis and HEp-2

835

bioassay, Anal. Bioanal. Chem. 2013, 405, pp. 191–201.

To

Monitor

Tyrosine-Induced

Metabolome

41 ACS Paragon Plus Environment

Alterations

in

Journal of Agricultural and Food Chemistry

836

(34) Stark, T. D.; Losch, S.; Wakamatsu, J.; Balemba, O. B.; Frank, O.; Hofmann, T.

837

UPLC-ESI-TOF MS-Based Metabolite Profiling of the Antioxidative Food Supplement

838

Garcinia buchananii, J. Agric. Food Chem. 2015, 63, pp. 7169–7179.

839

(35) Frank, O.; Kreissl, J. K.; Daschner, A.; Hofmann, T. Accurate determination of

840

reference materials and natural isolates by means of quantitative (1)h NMR

841

spectroscopy, J. Agric. Food Chem. 2014, 62, pp. 2506–2515.

842

(36) Monakhova, Y. B.; Ruge, W.; Kuballa, T.; Ilse, M.; Winkelmann, O.; Diehl, B.;

843

Thomas, F.; Lachenmeier, D. W. Rapid approach to identify the presence of Arabica

844

and Robusta species in coffee using 1H NMR spectroscopy, Food Chem. 2015, 182,

845

pp. 178–184.

846

(37) Monakhova, Y. B.; Kuballa, T.; Lachenmeier, D. W. Chemometric methods in

847

NMR spectroscopic analysis of food products, J. Anal. Chem. 2013, 68, pp. 755–766.

848

(38) Sousa, S.; Magalhães, A.; Ferreira, M. M. C. Optimized bucketing for NMR

849

spectra - Three case studies, Chemometrics Intell. Lab. Sys. 2013, 122, pp. 93–102.

850

(39) French, G. L.; Ling, J.; Hui, Y.-W.; Oo, H. K. T. Determination of methicillin-

851

resistance in Staphylococcus aureus by agar dilution and disc diffusion methods, J.

852

Antimicrob. Chemother. 1987, 20, pp. 599–608.

853

(40) Cockerill, F. Methods for dilution antimicrobial susceptibility tests for bacteria that

854

grow aerobically. Approved standard; Clinical and Laboratory Standards Institute:

855

Wayne, Pa., 2015.

856

(41) Fenn, P.; Durbin, R. D.; Kuntz, J. E. Conversion of tryptophan to indole-3-acetic

857

acidand other metabolites by Cerafocysfis fagacearum, Physiol. Plant Pathol. 1978,

858

12, pp. 297–309.

42 ACS Paragon Plus Environment

Page 42 of 46

Page 43 of 46

Journal of Agricultural and Food Chemistry

859

(42) Vleggaar, R.; Wessels, P. L. Stereochemistry of the Dehydrogenation of (2S)-

860

Histidine in the Biosynthesis of Roquefortine and Oxaline, J. C. S. Chem. Commun.

861

1980, 4, pp. 160–162.

862

(43) Ries, M. I.; Ali, H.; Lankhorst, P. P.; Hankemeier, T.; Bovenberg, R. A. L.;

863

Driessen, A. J. M.; Vreeken, R. J. Novel key metabolites reveal further branching of

864

the roquefortine/meleagrin biosynthetic pathway, J. Biol. Chem. 2013, 288,

865

pp. 37289–37295.

866

(44) O'Brien, M.; Nielsen, K. F.; O'Kiely, P.; Forristal, P. D.; Fuller, H. T.; Frisvad, J.

867

C. Mycotoxins and other secondary metabolites produced in vitro by Penicillium

868

paneum Frisvad and Penicillium roqueforti Thom isolated from baled grass silage in

869

Ireland, J. Agric. Food Chem. 2006, 54, pp. 9268–9276.

870

(45) Bertinetti, V.; Pen N. I.; Cabrera G. M. An antifungal tetrapeptide from the culture

871

of Penicillium canescens, Chem. Biodivers. 2009, 6, pp. 1178–1184.

872

(46) Karplus, M. Contact electron-spin coupling of nuclear magnetic moments, J.

873

Chem. Phys. 1959, 30, pp. 11–15.

874

(47) Karplus, M. Vicinal Proton Coupling in Nuclear Magnetic Resonance, J. Am.

875

Chem. Soc. 1963, 85, pp. 2870–2871.

876

(48) Minch, M. J. Orientational dependence of vicinal proton-proton NMR coupling

877

constants: The Karplus relationship, Concepts Magn. Reson. 1994, 6, pp. 41–56.

878

(49) Kawahara, N.; Nozawa, K.; Nakajima, S.; Kawai, K. Studies on Fungal Products.

879

Part 13.' Isolation and Structures of Dithiosilvatin and Silvathione, Novel

880

Dioxopiperazine Derivatives from Aspergillus silvaticus, J. Chem. Soc. Perkin Trans.

881

I. 1987, 9, pp. 2099–2101.

43 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

882

(50) Usami, Y.; Aoki, S.; Hara, T.; Numat, A. New Dioxopiperazine metabolites from

883

a Fusarium species separated from a marine alga, J. Antibiot. 2002, 55, pp. 655–659.

884

(51) Yang, M.-H.; Li, T.-X.; Wang, Y.; Liu, R.-H.; Luo, J.; Kong, L.-Y. Antimicrobial

885

metabolites from the plant endophytic fungus Penicillium sp, Fitoterapia. 2017, 116,

886

pp. 72–76.

887

(52) Capon, R. J.; Stewart, M.; Ratnayake, R.; Lacey, E.; Gill, J. H. Citromycetins and

888

bilains A-C: new aromatic polyketides and diketopiperazines from Australian marine-

889

derived and terrestrial Penicillium spp, J. Nat. Prod. 2007, 70, pp. 1746–1752.

890

(53) Burns, K. L.; Oldham, C. D.; Thompson, J. R.; Lubarsky, M.; May, S. W. Analysis

891

of the in vitro biocatalytic production of poly-(β)-hydroxybutyric acid, Enzyme Microb.

892

Technol. 2007, 41, pp. 591–599.

893

(54) Kopp-Holtwiesche, B.; Rehm, H. J. Antimicrobial action of roquefortine, J.

894

Environ. Pathol. Toxicol. Oncol. 1990, 10, pp. 41–44.

895

(55) Yang, H.; Li, F.; Ji, N. Alkaloids from an algicolous strain of Talaromyces sp,

896

Chin. J. Ocean. Limnol. 2016, 34, pp. 367–371.

897

(56) Narui, K.; Noguchi, N.; Saito, A.; Kakimi, N.; Motomura, N.; Kubo, K.; Takamoto,

898

S.; Sasatsu, M. Anti-infectious Activity of Tryptophan Metabolites in the L-

899

Tryptophan– L-Kynurenine Pathway, Biol. Pharm. Bull. 2009, 32, pp. 41–44.

900

(57) Walia, R.; Dhamija, K.; Akhtar, Vandana, Md. J.; Lamba, H. S. Synthesis of novel

901

substituted benzimidazole derivatives as potential antimicrobial agents, J. Pharm.

902

Chem. Biol. Sci. 2012, 2, pp. 293–298.

903

(58) da Rocha Neto, A. C.; Maraschin, M.; Di Piero, R. M. Antifungal activity of

904

salicylic acid against Penicillium expansum and its possible mechanisms of action,

905

Int. J. Food Microbiol. 2015, 215, pp. 64–70.

44 ACS Paragon Plus Environment

Page 44 of 46

Page 45 of 46

Journal of Agricultural and Food Chemistry

906

(59) Drozdenko, N. P.; Shcherbankov, L. A. Chemical preservation of clover, Khim.

907

Sel'sk. Khoz. 1968, 6, pp. 457–459.

908

(60) Yoshimura H.; Sakai T.; Kuwahara Y.; Ito M.; Tsuritani K.; Hirasawa Y.;

909

Nagamatsu T. Effects of kynurenine metabolites on mesangial cell proliferation and

910

gene expression, Exp. Mol. Pathol. 2009, 87, pp. 70–75.

911

(61) Lucca de, A. J.; Bland, J. M.; Vigo, C. B.; Jacks, T. J.; Peter, J.; Walsh, T. J. D-

912

Cecropin B: proteolytic resistance, lethality for pathogenic fungi and binding

913

properties, Med. Mycol. 2000, 38, pp. 301–308.

914

(62) Fleming, A. On the antibacterial action of cultures of a Penicillium, with special

915

reference to their use in the isolation of B. influenze, Br. J. Exp. Pathol. 1929, 10,

916

pp. 226–236.

917

(63) Marxen, S.; Stark, T. D.; Rütschle, A.; Lücking, G.; Frenzel, E.; Scherer, S.;

918

Ehling-Schulz, M.; Hofmann, T. Multiparametric Quantitation of the Bacillus cereus

919

Toxins Cereulide and Isocereulides A-G in Foods, J. Agric. Food Chem. 2015, 63,

920

pp. 8307–8313.

921

(64) Anderson, B. E.; Grove, M. Peptides comprising aromatic D-amino acids and

922

methods of use, United States Patent Application Publication. 2010.

45 ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

206x138mm (150 x 150 DPI)

ACS Paragon Plus Environment

Page 46 of 46