Mechanism of Arachidonic Acid Accumulation during Aging in

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Mechanism of Arachidonic Acid Accumulation During Aging in Mortierella alpina: A Large-Scale Label-Free Comparative Proteomics Study Yadong Yu, Tao Li, Na Wu, Lujing Ren, Ling Jiang, Xiao-Jun Ji, and He Huang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03284 • Publication Date (Web): 25 Oct 2016 Downloaded from http://pubs.acs.org on October 27, 2016

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Journal of Agricultural and Food Chemistry

Mechanism of Arachidonic Acid Accumulation During Aging in Mortierella alpina: A Large-Scale Label-Free Comparative Proteomics Study Yadong Yu1,#, Tao Li2,#, Na Wu2, Lujing Ren1,2, Ling Jiang3,*, Xiaojun Ji1,2,*, He Huang4,5,* 1

Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University, Nanjing, 211800

2

College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, 211800 3

College of Food Science and Light Industry, Nanjing Tech University, Nanjing, 211800 4

5

School of Pharmaceutical Sciences, Nanjing Tech University, Nanjing, 211800

State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing, 211800

#

These authors contributed equally to this work.

*

Corresponding authors:

Ling Jiang, Ph.D Associated Professor College of Food Science and Light Industry, Nanjing Tech University No.30 Puzhu South Road, Nanjing, 211800, China Tel: (86) 25-58139942 Email: [email protected] Xiaojun Ji, Ph.D Associated Professor

ACS Paragon Plus Environment


Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University College of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University No.30 Puzhu South Road, Nanjing, 211800, China Tel: (86) 25-58139942 Email: [email protected] He Huang, Ph.D. Professor School of Pharmaceutical Sciences, Nanjing Tech University State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University No.30 Puzhu South Road, Nanjing, 211800, China Tel: (86) 25-58139942 Email: [email protected]


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1

ABSTRACT: Arachidonic acid (ARA) is an important polyunsaturated

2

fatty acid, which has various beneficial physiological effects on the

3

human body. The aging of Mortierella alpina (M. alpina) has long been

4

known to significantly improve ARA yield, but the exact mechanism is

5

still elusive. Herein, multiple approaches including large-scale label-free

6

comparative proteomics were employed to systematically investigate the

7

mechanism mentioned above. Upon ultrastructural observation, abnormal

8

mitochondria were found to aggregate around shrunken lipid droplets.

9

Proteomics analysis revealed a total of 171 proteins with significant

10

alterations of expression during aging. Pathway analysis suggested that

11

reactive oxygen species (ROS) were accumulated and stimulated the

12

activation of the malate/pyruvate cycle and isocitrate dehydrogenase,

13

which

14

EC:4.2.1.17-hydratase might be a key player in ARA accumulation during

15

aging. These findings provide a valuable resource for efforts to further

16

improve the ARA content in the oil produced by aging M. alpina.

17

KEYWORDS:

18

mechanism, Mortierella alpina

might

provide

aging,

additional

arachidonic

NADPH

acid,

for

ARA synthesis.

proteomics,

19


molecular


20

INTRODUCTION

21

Arachidonic acid (ω-6, 5, 8, 11, 14-cis-eicosatetraenoic acid; ARA), an

22

important polyunsaturated fatty acid, has broad applications in a number

23

of different fields such as medicine, cosmetics, food industry and

24

agriculture.1-4 Microbial oils have long been suggested as an alternative

25

source of ARA and an increasing body of work has focused on effective

26

ARA production by microbial fermentation.5 Mrotierella alpina (M.

27

alpina), a filamentous fungus, is a prominent producer of ARA-rich oil.1, 5

28

Various strategies such as genetic modification or nutritional and

29

morphological control, have been developed to efficiently produce

30

ARA-rich oil. Aging technology, which employs a cell culture step under

31

carbon source limitation following regular fermentation, was shown to be

32

an effective way to improve ARA content in M. alpina.5 For example,

33

Streekstra et al. developed a two-stage culture strategy to improve ARA

34

production based on aging technology. When glucose was completely

35

consumed in the second stage, the ARA content of the lipids increased,

36

reaching up to 60%.6 In our previous work, we proposed a new method

37

which employed the addition of ethanol or KNO3 during aging. Under

38

optimal conditions, we found that the yield was 1.55 times higher than

39

with traditional aging technology.7

40

While different aging protocols have been developed to improve

41

ARA accumulation, attempts to uncover the exact mechanism by which


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the ARA content increases during the aging process are still in their

43

infancy. A better understanding of the mechanism can pave the way for

44

developing new aging protocols or even entirely new strategies for ARA

45

production.

46

Recently, proteomics methods have become a powerful tool to

47

investigate complex cellular events and molecular mechanisms in

48

microorganisms.8 Using a metabolomics approach, we found that the

49

ARA content was not only increased at the expense of the other fatty

50

acids, which were degraded, but also due to the biosynthesis of additional

51

ARA during aging.9 However, the way in which enzymes and other

52

proteins participate in ARA accumulation during M. alpina aging remains

53

unexplored.

54

In this work we used label-free quantitative proteomics methods and

55

bioinformatics tools to identify proteins with significantly changed

56

expression profiles, as well as to glean their functions during the aging

57

process of M. alpina. Ultimately, a number of key proteins and pathways

58

that account for a bulk of the ARA accumulation observed during the M.

59

alpina aging process have been revealed.

60

MATERIALS AND METHODS

61

Microorganism and medium

62

M. alpina R807 (CCTCC M2012118) used in this study was obtained

63

from the China Centre for Type Culture Collection.10 The PDA medium



64

consisted of 25 g/L glucose, 200 g/L potato extract and 20 g/L agar. The

65

seed medium consisted of 30 g/L glucose, 3 g/L NaNO3, 3 g/L KH2PO4, 6

66

g/L yeast extract and 0.5 g/L MgSO4.7H2O. The fermentation medium

67

consisted of 4 g/L KH2PO4, 3 g/L NaNO3, 0.6 g/L MgSO4.7H2O, 80 g/L

68

glucose and 10 g/L yeast extract.

69

Culture and aging conditions

70

The culture and aging conditions for M. alpina were the same as reported

71

in our previous work.9 In short, a loop was used to transfer a small

72

amount of M. alpina mycelium from a seed tube (containing 30%

73

glycerol and stored at -80℃) to PDA medium, followed by incubation in

74

an electro-thermal incubator at 25°C. After 7 days of incubation, the PDA

75

medium surface covered with white mycelium and was cut into square

76

pieces (1 cm× 1 cm) using a sterile shovel. Two square pieces were

77

transferred individually into 500 mL flasks containing 100 mL seed

78

medium. Seed culture was conducted for 24 h after which 10% (v/v) of

79

the culture broth was used to inoculate the fermentation broth. 500 mL

80

baffled flasks containing 100 mL of fresh fermentation medium were

81

used for fermentations. After a course of regular fermentation, mycelia

82

were continuously cultured without carbon source for the aging process.

83

All cultivations were carried out at 25℃, with an initial pH of 6.0 under

84

constant orbital shaking at 125 rpm.

85

Dry cell weight, total lipid, fatty acid profile and media component


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analysis

87

Glucose concentration was analyzed using a SBA-40C glucose oxidase

88

electrode (Shandong Academy of Sciences, China). To determine dry cell

89

weight (DCW), the mycelia were harvested and separated by filtration

90

through a conventional filter paper. Subsequently, the mycelia were

91

washed three times and dried at 65 ℃ to constant weight (approx. 8h).

92

Lipid extraction and fatty acid analysis was carried out according to our

93

previously reported methods.11, 12 In short, the dried mycelia were

94

smashed into a powder in a mortar. Subsequently, 150 mL

95

methanol/chloroform (1:2, v/v) were used to extract the lipids from 2 g of

96

the resulting powder in a Soxhlet extractor for 8 h at 75 °C. The resulting

97

solvent was evaporated and recycled. For fatty acid analysis, a GC system

98

(GC-2010, Shimadzu, Japan) containing a flame ionization detector (FID)

99

and a capillary column (DB-23, 60 m × 0.22 mm, Agilent, USA) was

100

employed. The column was heated from 100 °C to 196 °C at 25 °C/min

101

and subsequently raised to 220 °C at 2 °C /min. Finally, the column was

102

maintained at 220 °C for 6 min. The temperature of the FID detector was

103

set as 280 °C. N2 was used as carrier gas. The injector was kept at 250 °C

104

and the inject volume was 1 µL. The contents of amino acids in the

105

fermentation medium was analyzed by the Physical and Chemical Testing

106

Center of Jiangsu Province (Nanjing, China) using a Hitachi L-8800

107

amino acid analyzer. The contents of citric , malic , lactic , and succinic



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acids, as well as ethanol in the fermentation media was analyzed by the

109

Gratech Company (Shanghai, China) using a high performance liquid

110

chromatography system (1260, Agilent, USA).

111

Confocal

112

microscopy

113

Confocal fluorescence microscopy was conducted according to a

114

previously reported method.13 Briefly, the mycelia were collected by

115

centrifugation and resuspended in glycerol to a final concentration of 0.1

116

g/mL. 5 µL Nile Red (J & K Scientific, China) stock solution (0.4 mg/mL)

117

was added to 3 mL of the mycelia-glycerol suspension and mixed by

118

gentle shaking for 1 min. After incubation in darkness for 5-10 min at

119

room temperature, samples were directly used for fluorescence-activated

120

cell sorter (FACS) and microscopic analyses. The fluorescence excitation

121

wavelength was set at 488 nm and the emission wavelength scanning

122

range was set from 500 nm to 750 nm.

123

fluorescence

microscopy

and

transmission

electron

For transmission electron microscopy (TEM) observation, mycelia

124

were cut into small pieces and fixed in 2.5% (w/v) glutaraldehyde in PBS

125

(pH 7.4) at 4°C for two days. To remove the glutaraldehyde, the samples

126

were washed in three 15 min steps with 0.1 M PBS (pH7.4).

127

Subsequently, the samples were additionally fixed with 2% (w/v) osmium

128

tetroxide (EM grade, Nakalai Tesque, Japan) at room temperature for 1 h.

129

The stained samples were dehydrated by serial rinses with ethanol in


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water solutions with concentrations of 35%, 50%, 70%, 90%, 95%,

131

respectively, in that order, followed by a final rinse in absolute ethanol.

132

After embedding in epoxy resin, samples were prepared by cutting 90 nm

133

sections using an EM UC6 Ultramicrotome (Leica, Germany). After

134

staining with uranyl acetate (EM grade, Electron Microscopy Sciences,

135

USA) and lead citrate (EM grade, Electron Microscopy Sciences, USA),

136

the sections were examined under a JEM-1011 transmission electron

137

microscope (JEOL, Japan).

138

Proteomics Experiments

139

Sample preparation

140

After harvesting by centrifugation at 5000 g for 3 min, the cell pellets

141

were resuspended on ice in 200 µL lysis buffer (4% SDS, 100 mM DTT,

142

150 mM Tris-HCl pH 8.0). Cells were disrupted using a Fastprep-24®

143

homogenizer at 700 bar for 3-4 cycles (MP Biomedical, USA), and the

144

lysates subsequently boiled for 5 min. The samples were further

145

homogenized by ultrasonication using a Scientz® ultrasonicator set to 20 %

146

duty cycle, 200 W power output, for 10 min and boiled again for another

147

5 min. After centrifugation at 14 000 g for 15 min, the supernatants were

148

collected and the total protein contents quantified using a BCA Protein

149

Assay Kit (Bio-Rad, USA) with 5 mg/mL BSA as a reference.

150

Protein digestion

151

Protein digestion was performed according to the method described by



152

Wisniewski et al.14 Briefly, low-molecular-weight components were

153

removed using 200 µL UA buffer (8 M Urea, 150 mM Tris-HCl pH 8.0)

154

by repeated ultrafiltration. 100 µL of a 0.05 M iodoacetamide solution in

155

UA buffer was added to the samples to block reduced cysteine residues

156

followed by incubation for 20 min in darkness. The filter was washed

157

three times with 100 µL UA buffer and twice with 100 µL 25 mM

158

NH4HCO3. The protein suspension was digested overnight at 37 °C by

159

adding 3 µg trypsin (Promega, USA) suspended in 40 µL 25 mM

160

NH4HCO3. The resulting peptide content was determined by measuring

161

the absorption at 280 nm.

162

Liquid Chromatography (LC)-Electrospray Ionization (ESI) Tandem

163

MS (MS/MS) analysis by Q Exactive

164

The peptides in each sample were desalted on C18 Cartridges (Sigma,

165

USA), concentrated by vacuum centrifugation and reconstituted in 40 µL

166

0.1% trifluoroacetic acid. MS experiments were carried out on a Q

167

Exactive mass spectrometer that was coupled to an Easy nLC (Thermo

168

Fisher Scientific, USA). An aliquot comprising 5 µg of peptides in buffer

169

A (2% acetonitrile and 0.1% formic acid) was loaded onto a C18-reversed

170

phase column (Thermo Scientific, USA) and separated with a linear

171

gradient of buffer B (80% acetonitrile and 0.1% formic acid). MS data

172

were obtained using a data-dependent top10 method choosing the most

173

abundant precursor ions from the survey by scanning dynamically for


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HCD fragmentation. The target value was determined based on predictive

175

Automatic Gain Control (pAGC). MS experiments were performed in

176

triplicate for each sample.

177

Bioinformatics analysis

178

Sequence database search and data analysis

179

The MS data were analyzed using MaxQuant software (version 1.3.0.5).

180

MS data were searched against the UniProtKB (uniprot_fungi incertae

181

sedis_752077_20160118.fasta, 752077 total entries). An initial search

182

was conducted at a precursor mass window of 6 ppm. The search

183

followed the enzymatic cleavage rule of Trypsin/P and allowed a

184

maximum of two missed cleavage sites. Carbamidomethylation of

185

cysteines was defined as fixed modification and protein N-terminal

186

acetylation and methionine oxidation were defined as variable

187

modifications. The cutoff of global false discovery rate (FDR) for peptide

188

and protein identification was set to 0.01. Label-free quantification was

189

performed in MaxQuant as previously described.15 The sequence data of

190

the target proteins was retrieved from the UniProtKB database in batches.

191

The retrieved sequences were searched against the SwissProt database

192

using the NCBI BLAST+ client software version 2.2.28. The top 10 blast

193

hits with E-values of less than 1e-3 for each query sequence were

194

retrieved and loaded into Blast2GO (version 2.7.2) for GO annotation and

195

KEGG pathway analysis.16-18



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196

qRT-PCR analysis

197

The M. alpina mycelia were harvested from 2 mL culture broth aliquots,

198

immediately frozen in liquid nitrogen and stored at -80 ℃ for further

199

analysis. Total RNA was extracted using TRizol solution (Invitrogen,

200

USA) and the RNA concentration measured using a Hofer MV-25

201

spectrophotometer

202

electrophoresis was employed to assess RNA integrity. RNA samples

203

were subsequently treated with DNase (Zoonbio, China). Reverse

204

transcriptase reactions were performed using 2.5 µg of total RNA, Oligo

205

dT and random primers (Zoonbio, China) to obtain cDNA. A LightCycler

206

3.0 system (Roche, USA) and SYBR Green Qpcr Mix (Zoonbio, China)

207

were used for RT-qPCR experiments and 2−△△Ct analysis. 18S rRNA

208

was used as internal reference. All primer sequences are listed in Table

209

S1.

210

Enzyme activity assay

211

Malic enzyme (ME) and isocitrate dehydrogenase (IDH) activity assays

212

were carried out as described previously.19, 20 In short, ME activity was

213

determined using an assay system containing 0.069 M Tris-HCl (pH 7.4),

214

0.004 M MgCl2, 1.034 mM malate and 0.234 mM NADPNa2

215

(minimum >97%, BIOSHARP, China). Isocitrate dehydrogenase activity

216

was

217

KH2PO4-K2HPO4 buffer (pH 7.0), 0.004 M MgCl2, 0.0002 M

determined

(Amersham

using

an

Pharmacia,

assay

USA).

Agarose

system containing


0.037

gel

M

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218

DD-isocitrate (J & K Scientific, China) and 0.0002 M NADPNa2

219

(BIOSHARP, China). The concentrations given for the chemicals used for

220

the enzymatic activity assays are the final concentrations in the reaction

221

mixture. All enzyme activities were measured using continuous

222

spectrophotometric assays to monitor the oxidation or reduction of

223

NADPH at 340 nm. One unit of enzyme activity was defined as the

224

amount of enzyme that catalyzes the conversion of 1 µM of NADP(H) in

225

1 min at room temperature (22°C).

226

ROS detection

227

The contents of reactive oxygen species (ROS) in M. alpina mycelia was

228

determined according to a previously published method,21 with minor

229

modifications as follows: The M. alpina mycelia in a 20 mL aliquot of

230

culture

231

2’,7’-dichlorouorescein diacetate solution (DCFH-DA, Sigma, USA), and

232

after thorough incorporation of DCFH-DA, the mycelia were further

233

cultured at 30°C for another 30 min under constant orbital shaking at 125

234

rpm. The stained mycelia were subsequently harvested, washed twice

235

with PBS buffer, and the fluorescence intensity (FLU) measured on a

236

fluorescence microplate reader (Molecular Devices, USA), with the

237

excitation wavelength set to 488 nm, and emission wavelength at 520 nm.

238

FLU was divided by the dry cell weight (DCW) of the examined mycelia

239

to obtain the relative fluorescence density (RFLU).

broth

were

stained

with

5

µL


of

a

10

mg/mL


240

Statistical Analysis

241

Statistical analysis was performed with a one-way ANOVA in Origin 6.1

242

software. Results were accepted as statistically significant at a 0.05

243

significance level.

244

RESULTS AND DISCUSSION

245

Changes of biomass, lipid accumulation, fatty acid profile and media

246

components during the aging process

247

As shown in Fig. 1A, after 156 h of fermentation, M. alpina mycelia

248

entered the aging period after glucose was exhausted. The biomass and

249

lipid concentration increased to 28.2 g/L and 11.2 g/L, respectively, at the

250

end of regular fermentation, but decreased to 23.8 g/L and 9.4 g/L during

251

the aging process. We further analyzed the percentage of different fatty

252

acids in total lipids and the concentration of each fatty acid during the

253

aging process. We found that the ARA percentage increased from 37.2%

254

to 62.0% and ARA concentration increased from 3.9 g/L to 5.8 g/L.

255

However, both the percentages and the concentrations of the other fatty

256

acids (C16:0, C18:0, C18:1, C18:2, C18:3, C20:3) were decreased (Fig.

257

1B-C). Notably, the percentage and concentration of the saturated fatty

258

acids (C16:0, C18:0) decreased much more than those of unsaturated

259

ones (C18:2, C18:3, C20:3). In agreement with our previous findings,9

260

these results suggested that the other fatty acids were used as precursors

261

for the biosynthesis of additional ARA.


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Nitrogen and carbon sources in the media, other than glucose, may

263

also affect the microbial oil synthesis. Therefore, we further quantified

264

the contents of amino acids, some organic acids and ethanol in the media

265

during the aging process (Table S2-3). As shown in Table S2, the total

266

amount of free amino acids decreased slightly, which might suggest that

267

mycelia can still absorb a certain amount of amino acids to fulfil basic

268

biological functions as well as ARA biosynthesis. This conclusion might

269

be further supported by the observed decrease of the concentrations of

270

some amino acids. such as phenylalanine (Phe), since it has been reported

271

that M. alpina can utilize Phe by the phenylalanine-hydroxylating system,

272

to provide NADPH and acetyl-CoA for lipid accumulation.22 Interestingly,

273

there were six amino acids, valine (Val) and tyrosine (Tyr) being chief

274

among them, whose contents slightly increased during the aging process.

275

This phenomenon might be a consequence of protein degradation in the

276

aged mycelia, since cell aging can lead to protein oxidation and

277

proteolysis in eukaryotes.23 Furthermore, some amino acids such as Lys

278

can be catabolized by oleaginous fungi to synthesize acetyl-CoA, which

279

is a critical substrate for microbial oil synthesis.24 Thus, it can be

280

reasoned that M. alpina degraded more proteins to obtain Lys, which may

281

lead to the increase of Lys in the media. The contents of the other amino

282

acids, such as threonine (Thr) and aspartic acid (Asp) were relatively

283

stable or they were undetectable in the media, which suggests that the



284

uptake or secretion rates of these amino acids were not significantly

285

changed.

286

The contents of other carbon sources including citric acid, malic

287

acid, lactic acid, succinic acid and ethanol were also determined. We

288

found that all these carbon sources were undetectable in the media (Table

289

S3). These results indicated that, during the aging process, the carbon

290

sources such as succinic acid and ethanol were scarce for M. alpina and

291

the secretion of these compounds from mycelia might also be very

292

limited under our experimental conditions. When glucose and other

293

extracellular carbon sources were exhausted, the stored lipids started to

294

be degraded to provide essential energy for the cells.25, 26 Consistently

295

with these findings, we also found that many fatty acids decreased

296

significantly, whereby ARA was an exception (Fig.1C). Actually,

297

although the in-depth mechanism is still unclear, supplying an additional

298

carbon source such as ethanol is helpful for increased synthesis of ARA

299

during the aging process of M. alpina. We believe it will be exciting and

300

meaningful to explore the influence of supplying organic acids, such as

301

malic acid, on the ARA synthesis during M. alpina aging, since these

302

organic acids are pivotal in lipid biosynthesis.25

303

Changes of mycelial morphology and ultrastructure during the aging

304

process

305

Microbial morphology and ultrastructure are intimately linked to the


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physiological status of the cells. In order to further investigate how the

307

morphology and ultrastructure of the mycelia changed during the aging

308

process, confocal microscopy and TEM were employed.

309

As depicted in Fig.2A, at the end of regular fermentation process

310

(156h), most of the M. alpina mycelia showed an unbroken, filamentous

311

appearance. A number of red-colored spheres were visible in the mycelia

312

(Fig.2A). A large proportion of the cell volume was occupied by lipid

313

droplets (LDs) and some of the LDs were in the process of fusing or

314

budding. More than one nucleus was found per cell body, which is

315

expected since M. alpina is a multinucleate fungus (Fig.2B).27 At the

316

middle stage of the aging process (192 h), most of the mycelia were still

317

unbroken. But LDs in some parts of the mycelia disappeared or became

318

smaller (highlighted in Fig.2C with blue arrows). TEM observation

319

further confirmed that LDs had shrunk. Interestingly, mitochondria were

320

found to aggregate around the shrunken LDs (Fig.2D). As we know, when

321

energy supply is insufficient, triacylglycerides (TAG) stored in the lipid

322

droplets can be hydrolyzed to supply fatty acids which then enter

323

mitochondria where they are used to produce ATP via beta-oxidation and

324

oxidative phosphorylation. It has also been observed that mitochondria

325

will relocate closer to and interact with LDs more actively when the

326

energy supply or fatty acid pool of the cells is insufficient.28 Therefore,

327

based on these findings, we hypothesized that when mycelia lacked



328

sufficient external carbon sources and were experiencing a shortage of

329

energy, mitochondria would relocate to the close proximity of LDs to

330

utilize the stored TAG, which finally led to the shrinkage of LDs. In

331

addition, the mitochondria seemed to be enlarged and mitochondrial

332

cristae became less pronounced (Fig.2D), suggesting that aged

333

mitochondria might be malfunctioning. This is in agreement with the

334

findings reported for the aging processes in other filamentous fungi.29, 30

335

At the end of the aging process (240 h), both the number and the size of

336

LDs decreased significantly. The mycelia had an irregular appearance and

337

some of them seemed to be broken (Fig.2E-F), suggesting that M. alpina

338

mycelia had begun to decompose at the end of the aging process.

339

Comparative proteomics analysis of M. alpina during the aging

340

process

341

The percentage and concentration of ARA increased markedly in the

342

middle stage of aging process (192 h) (Fig. 1B-C), suggesting that the

343

physiological status of the M. alpina mycelia was changed significantly at

344

this time point. Moreover, at the end of the aging process (240 h),

345

mycelia began to decompose and cytoplasm likely leaked out. We thus

346

analyzed the protein expression profiles of mycelia from the middle stage

347

of the aging process (192 h) and compared them to those of mycelia from

348

the end of the regular fermentation, before entering aging (156 h). For

349

proteomics experiments, the middle stage of the aging process (192 h)


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and the end of the regular fermentation (156 h) were designated as aging

351

group and control group, respectively.

352

Cluster analysis of differentially expressed proteins

353

SDS-PAGE results indicated that the as-prepared protein samples were of

354

adequate quality for the following proteomics experiments (Fig.3A). By

355

using the large-scale label-free comparative proteomics methodology and

356

statistical filtration (fold change>2, p

Mechanism of Arachidonic Acid Accumulation during Aging in

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