Metabolomics Reveal Optimal Grain ... - ACS Publications

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Article

Metabolomics reveal optimal grain preprocessing (milling) toward rice koji fermentation Sunmin Lee, Da Eun Lee, Digar Singh, and Choong Hwan Lee J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05131 • Publication Date (Web): 02 Mar 2018 Downloaded from http://pubs.acs.org on March 3, 2018

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.

Journal of Agricultural and Food Chemistry 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 33

Journal of Agricultural and Food Chemistry

Metabolomics reveal optimal grain pre-processing (milling) toward rice koji fermentation Sunmin Lee, Da Eun Lee, Digar Singh, Choong Hwan Lee* Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea; [email protected]

(S.L.);

[email protected]

(D.E.L);

[email protected]

(D.S.);

[email protected] (C.H.L)

*Corresponding author Choong Hwan Lee Department of Bioscience and Biotechnology, Konkuk University, Seoul 05029, Republic of Korea Tel.: +82-2-2049-6177; Fax: +82-2-455-4291; E-mail address: [email protected]

1

ACS Paragon Plus Environment


1

ABSTRACT

2

A time-correlated mass spectrometry (MS) based metabolic profiling was performed for

3

rice koji made using the substrates with varying degrees of milling (DOM). Overall, 67

4

primary and secondary metabolites were observed as significantly discriminant among

5

different samples. Notably, a higher abundance of carbohydrate (sugars, sugar alcohols,

6

organic acids, phenolic acids) and lipid (fatty acids, lysophospholipids) derived metabolites

7

with enhanced hydrolytic enzyme activities were observed for koji made with substrate's

8

DOM 5–7, at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid)

9

were relatively higher in koji with substrate's DOM 0, followed by DOM 5 > 7 > 9 and 11, at

10

96 h. Hence, we conjecture that the rice substrate pre-processing between DOM 5-7 was

11

potentially optimal toward koji fermentation with end-product being rich in distinctive

12

organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate pre-

13

processing steps vital for commercial koji making.

14

KEYWORDS: Rice Koji, Degree of milling, Fermentation, Mass spectrometry, Enzyme

15

activity

2


Page 2 of 33

Page 3 of 33


16

1. INTRODUCTION

17

Fermentation, a customary food processing and preserving method, can be traced back to

18

thousands of years in pre-historic era.1 Considering the modern perspectives, the fermented

19

food products are globally relished as functional foods with the capacity to function as health

20

aids.2 Typically, a food fermentative bioprocess is carried out using diverse microflora

21

including fungi, yeast, and bacteria to improve the nutritional value and food safety with

22

enhanced organoleptic properties.3,4

23

The koji, being an inextricable component or starter for numerous fermented foods,

24

condiments, and beverages typical to East Asia, employed largely in food fermentative

25

bioprocesses.5 Koji is made through solid-state fermentation by inoculating steamed rice

26

grains with molds (A. oryzae), which germinates to secrete hydrolytic enzymes triggering

27

fermentation.6 Since nearly two millennia, Aspergillus strains have been used for the koji

28

fermentation, employed typically for rice wines (makgeoli, sake), soy sauce (ganjang, shoyu),

29

fermented soybean (doenjang, miso), soy-pepper paste (gochujang), and distilled spirits

30

(shochu).7 Particularly, rice koji fermentation with Aspergillus oryzae primarily occurs

31

through carbohydrate metabolism by secreted hydrolytic enzymes.8 In particular, the

32

hydrolytic enzymes secreted by A. oryzae (koji mold), modulates substrate textures through

33

affecting the release of metabolic gamut such as mono-sugars, amino acids, and fatty acids.9

34

In the fermentation process of any product, the starting substrate and inoculum are critical

35

to the quality of fermentation end-products. Previously, we correlated structural contours and

36

metabolic compositions of different rice varieties with culture growth, secreted enzymes as

37

well as metabolites in koji.10 Yoshizaki et al. have also reported the effects of three types of

38

rice koji (yellow, white, and black) on the volatile aroma compounds using gas

39

chromatography-mass spectrometry (GC-MS) based profiling.11 Functionally, the rice is a 3



40

valuable and rich source of antioxidants including phenolic compounds and has a hard husk

41

(hull) that protects the endosperm. Most of these compounds are distributed in different

42

portions of rice grain, particularly accessible through milling process. The commercial rice-

43

milling process generates products like husk and bran (pericarp) with low-value fractions.

44

After the husk is removed, the remainder is known as brown rice, which includes the bran,

45

embryo, and endosperm.12 Brown rice retains its bran layer and embryo bud, while the bran

46

and embryo of white rice are removed during complete milling. Liu et al. have highlighted

47

the effects of varying degrees of milling (DOM) on total flavonoid and phenolic

48

compositions as well as the associated antioxidant activities for brown rice .13 Determining an

49

appropriate DOM is vital for improving nutrient utilization and controlling potential hazards

50

associated with anti-nutritional factors to avoid nutrient loss due to over-milling.14

51

Metabolomics have increasingly been used to study the nutritional, functional, and

52

organoleptic aspects of fermented foods in a high-throughput manner owing to the

53

technological and computational advancements in mass spectrometry (MS) and related

54

databases, respectively. In recent years, the metabolomic perspectives of numerous fermented

55

foods viz., koji, doenjang, and gochujang, have been comprehensively studied to gain insights

56

of the corresponding nutritional, functional, and quality biomarkers.10,15,16 Herein, we

57

examined the subtle effects of rice substrate milling on metabolomic profiles and associated

58

biochemical phenotypes viz., enzyme activities and antioxidant phenotypes.

59 60

2. MATERIALS AND METHODS

61

2.1. Chemicals and Reagents. HPLC grade water, methanol, and acetonitrile were obtained

62

from Thermo Fisher Scientific (Waltham, MA, USA). Analytical grade chemicals viz., acetic

63

acid, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), casein 4


Page 4 of 33

Page 5 of 33


64

from bovine milk powder, Folin-Ciocalteu’s phenol, formaldehyde solution, formic acid,

65

methoxyamine hydrochloride, p-nitrophenol, p-nitrophenol β-D-glucopyranoside (p-NPG),

66

potassium persulfate, pyridine, sodium hydroxide, sodium acetate, starch, N-methyl-N-

67

(trimethylsilyl) trifluoroacetamide, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

68

(Trolox), and tyrosine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium

69

carbonate, sodium dihydrogen phosphate, and disodium hydrogen phosphate were purchased

70

from Junsei Chemical Co., Ltd. (Tokyo, Japan). Trichloroacetic acid was obtained from

71

Merck Millipore Co. (Darmstadt, Germany).

72

2.2. Rice Koji fermentation with substrates having varying DOM and sample harvest.

73

Brown rice samples of a Korean cultivar, Jinsang, were used in this study towards the

74

fermentative production of rice koji samples and differentially milled using a polishing

75

machine (Model MP-220, Yamamoto Co., Tokyo, Japan) to obtain rice samples with different

76

DOM. Each DOM was represented in terms of the proportion of embryo bud and bran layer,

77

remained in rice (Table 1). The 5 milled rice categories viz., DOM 0, DOM 5, DOM 7, DOM

78

9, DOM 11, were collected and stored at -20°C. The koji mold Aspergillus oryzae (KCCM

79

12698) procured from the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea),

80

was used as culture inoculum. A. oryzae was maintained on Malt Extract Agar (malt extract:

81

20 g, glucose: 20 g, peptone: 1 g, and agar: 20 g per liter) at 28°C. The fermentative

82

bioprocess steps of koji production were adapted from Lee et al.10 The fermented rice koji

83

samples were harvested at every 12 h (up to 96 h) and stored at deep freezing conditions (-

84

80°C) until analyses.

85

2.3. GC-TOF-MS Analysis. All sample extraction and derivatization steps were performed

86

as described by Lee at al.10 Gas chromatography time-of-flight MS (GC-TOF-MS) analysis

87

was performed on an Agilent 7890A GC system (Santa Clara, CA, USA) with a Pegasus HT 5



88

TOF-MS (Leco Corporation, St. Joseph, MI, USA). An RTx-5MS (30 m length × 0.25 mm

89

i.d., J & W Scientific, Folsom, CA, USA) was used a carrier gas (helium) at a constant flow

90

rate of 1.5 mL/min. The injector and ion source temperature were maintained at 250°C and

91

230°C, respectively. The oven temperature was maintained at 75°C for 2 min, and then

92

increased to 300°C at 15°C/min, which was sustained for 3 min. Next, 1 µL of sample was

93

injected with a mass scan range of 50–800 m/z. Overall, three samples as well as analytical

94

replicates were maintained for each variant.

95

2.4. LC-ESI-MS Analysis. The samples were extracted and analyzed for secondary

96

metabolites using ultra-high-performance liquid chromatography linear trap quadrupole ion

97

trap tandem mass spectrometry (UHPLC-LTQ-IT-MS/MS) and ultra-performance liquid

98

chromatography quadruple time-of-flight mass spectrometry (UPLC-Q-TOF-MS), using the

99

protocols described by Lee at al.10 Samples were separated on a syncronis C18 column with

100

100 mm × 2.1 mm, 1.7 µm particle size (Thermo Scientific). The mass spectra and

101

photodiode array range in both positive and negative ion mode were tuned for 100–1000 m/z

102

and 200-600 nm, respectively.

103

2.5. Data Processing and Multivariate Statistical Analysis. The raw datasets from GC-

104

TOF-MS and UHPLC-LTQ-IT-MS/MS analyses were transformed to netCDF (*.cdf) format

105

using Leco ChromaTOF and Thermo Xcalibur software, respectively. The respective netCDF

106

(*.cdf) files were subjected to MetAlign software (http://www.metalign.nl) mediated data

107

processing as described previously by Lee et al.10,

108

contained the suitable peak mass (m/z), retention times (min), and peak area information as

109

variables, was evaluated using SIMCA-P+ 12.0 software (Umetrics, Umea, Sweden) for

110

multivariate statistical (MVS) analysis. The data sets were log-transformed and unit variance

111

was scaled prior to principal component analysis (PCA) and partial least-squares discriminant

15

The resulting data matrix, which

6


Page 6 of 33

Page 7 of 33


112

analysis (PLS-DA) to compare the five rice koji. Significant differences (p-value < 0.05)

113

were tested by one-way analysis of variance using PASW Statistics 18 (SPSS, Inc., Chicago,

114

IL, USA). The putative metabolite identification was performed through matching respective

115

molecular weights and formula, retention time, mass fragmentation patterns, and UV

116

absorbance data available in literature and our in-house library (Table S1 and S2).

117

2.6. Enzyme Activities. The enzymatic activity assays including amylase, protease, β-

118

glucosidase were conducted according to the protocols described by Lee et al.10 Each rice koji

119

sample (10 g) in 90 mL of water was extracted by shaking in an incubator at 120 rpm and

120

30°C for 1 h. To evaluate enzymatic activities, filtered supernatants were used.

121

2.7. Determination of Antioxidant Activity. To determine the antioxidant activity of rice

122

koji samples, ABTS and FRAP assays were performed in triplicate. The ABTS antioxidant

123

assay was performed using the method partially adapted from Re et al. and Lee et al.10, 17 For

124

analysis, the ABTS stock solution was diluted using distilled water achieving a solution with

125

absorbance of 0.7 ± 0.02 at 750 nm. Each sample extract (20 µL) was incubated with ABTS

126

solution (180 µL) in 96-well plate, and the reaction was incubated at room temperature for 6

127

min under dark, and the absorbance was measured at 750 nm. The FRAP assay was

128

performed using a mixture of 300 mM acetate buffer (pH 3.6), 20 mM iron(III) chloride, and

129

10 mM TPTZ solution in 40 mM HCl (10:1:1, v:v:v). For analysis, 10 µL of sample was

130

added 300 µL of FRAP reagent and incubated at room temperature for 6 min. Absorbance

131

was measured at 570 nm. The results are represented as the Trolox equivalent antioxidant

132

capacity concentration (mM) per milligram of koji. The standard curves of Trolox ranged

133

from 0.0156 to 0.5 mM.

134

2.8. Physicochemical Characteristics. For evaluating physicochemical characteristics, 3 g

135

of koji samples were extracted with 30 mL of distilled water at 30°C for 1 h and 120 rpm. 7



136

The content of total sugar contents at 25°C expressed as °Brix was determined using a digital

137

refractometer (HI 96811, Hannah Instruments, Woonsocket, RI, USA). pH was measured

138

using a pH meter (Thermo). Titratable acidity was measured by titrating the samples with 0.1

139

N NaOH solution to a pH value of 8.4. The amino-type nitrogen contents were estimated by

140

adding 36% formaldehyde solution (20 mL) to the titrated samples, followed by sample re-

141

titration to pH 8.4 using 0.1 N NaOH solution. Amino-type nitrogen contents were

142

quantitatively estimated based on the titrated volume of 0.1 N NaOH and transformed to total

143

amino acid content.

144

3. RESULTS

145

3.1. Metabolic Profiling and Multivariate Analyses for Rice Koji fermented using the

146

Substrates with Varying DOMs. Differential metabolomes among the rice koji samples

147

made using the substrates with varying DOM were examined using multivariate analyses i.e.,

148

PCA and PLS-DA, based on GC-MS and LC-MS datasets. The PCA and PLS-DA model

149

based on rice koji samples according to the DOM are shown in Figure 1 and Supplementary

150

Figure 1, respectively. The principal component analysis (PCA) is an unsupervised,

151

multivariate statistical method for transforming an original set of correlated variables to a

152

new set of uncorrelated variables, called principal components (PC). On the other hand,

153

partial least squares discriminant analysis (PLS-DA) is commonly applied to evaluate the

154

clear distinctions between groups (variables) within the observed datasets.

155

The PCA score plot derived from GC-TOF-MS (Figure 1A) and UHPLC-LTQ-ESI-IT-

156

MS/MS (Figure B) data sets showed 51.2% (PC1: 39.8% and PC2: 11.4%) and 21.7% (PC1:

157

18.6% and PC2: 3.1%) variances, respectively. The PLS-DA model showed a similar pattern

158

of metabolic profiles compared to the PCA model indicating a steady alteration in rice koji

159

metabolomes during the course of fermentation. Intriguingly, each rice koji with different 8


Page 8 of 33

Page 9 of 33


160

DOM substrates exhibited different metabolite changes. However, similar patterns were

161

observed between rice koji with substrates having DOM 5 and 7 as well as DOM 9 and 11

162

samples.

163

Differential variables were selected based on variable importance in projection (VIP >1.0)

164

values and p-values (p < 0.05) obtained by PLS-DA model. The variable importance in

165

projection (VIP) values reflects the statistical significance of each variable in the model. A

166

total of 54 primary metabolites by GC-TOF-MS data (18 amino acids, 7 fatty acids, 11

167

organic acids, 15 sugars and sugar alcohols, and 4 others) were identified using standard

168

compounds, mass fragment patterns, of NIST and an in-house library (Table S1). Further, 13

169

secondary metabolites by UHPLC-ESI-LTQ-MS/MS data were selected as major compounds

170

related to the discrimination between rice koji with different DOM (Table S2). In particular,

171

apigenin-6-C-glucosyl-8-C-arabinoside, isovitexin-2''-O-glucoside, tricin-O-rutinoside, tricin,

172

pinellic acid, and 8 lysophospholipids including lysophosphatidylcholines (lysoPC) and

173

lysophosphatidylethanolamine (lysoPE) were identified by comparing the data to an in-house

174

library and published literature.

175

3.1.1 Temporal Primary Metabolomes for Rice Koji fermented using Substrates with

176

Varying DOMs. To visualize discriminative primary metabolites according to fermentation

177

time, a heat-map was made based on the GC-TOF-MS data. The trends of variations in

178

metabolite levels according to fermentation time are shown in Figure 2, with their relative

179

levels are represented as fold-changes normalized to the respective value of unfermented rice

180

with different DOM. The trends of sugar and sugar alcohol derivatives viz., adonitol, dulcitol,

181

gluconate, glycerate, maltose, myo-inositol, raffinose, sucrose, sorbitol, and xylose, were

182

gradually increased. The relative abundance of glucose was increased until 24 h and then

183

decreased until 96 h. In amino acid metabolism, the relative levels of serine, glycine, 9



184

isoleucine, threonine, valine, methionine, aspartate, alanine, phenylalanine, tryptophan, lysine,

185

gaba, ornithine, and pyroglutamate were increased, whereas those of asparagine and

186

glutamate were gradually decreased. Among the fatty acids, particularly palmitic acid,

187

linoleic acid, elaidic acid, oleic acid, linolenic acid, stearic acid, and myristic acid, were

188

increased. Metabolites related to the tricarboxylic acid (TCA) cycle such as citrate, malate,

189

fumarate, and succinate increased with fermentation time.

190

3.1.2 Relative Disparity in the Levels of Discriminant Metabolites in Rice Koji made

191

using the Substrates with Varying DOMs. To illuminate the relative levels of discriminant

192

metabolites among rice koji types made using the substrate with varying DOM representing

193

each fermentation time point, a heatmap was used for indicating the highest levels of

194

respective metabolites (Table 2). The comparison with substrates (unfermented rice) revealed

195

that most metabolites such as amino acids, organic acids, sugars and sugar alcohols and

196

secondary metabolites were relatively higher in DOM 0 koji, whereas fatty acids were highest

197

in DOM 5 koji. Depending upon the fermentation time point, the levels of metabolites in

198

various rice koji types changed from DOM 0 to DOM 5–7 at 24 h, and subsequently from

199

DOM 5–7 to DOM 7–9 at 36 h. Between 48–72 h, these metabolic trends changed in the rice

200

koji made with substrates having DOM 7–11. In contrast, the highest levels of amino acids,

201

sugars and sugar alcohols, nucleotide, fatty acids, and organic acids except secondary

202

metabolites were observed in DOM 0 rice koji samples at 96 h.

203

3.2 Variations in Enzymatic Production, Bioactivities, and Physiological Characteristics

204

for Rice Koji Types made using the Substrates with Varying DOM. The fermentation time

205

correlated enzymatic activities for different rice koji types made using the substrates with

206

varying DOM were determined to estimate the differential secretion of hydrolytic enzymes

207

by A. oryzae (Figure 3 A–C). In general, the amylase, protease, and β-glucosidase activities 10


Page 10 of 33

Page 11 of 33


208

were increased linearly with fermentation time in different rice koji types. The protease and

209

β-glucosidase activities were highest in the koji samples with DOM 7 substrates, whereas

210

amylase activity showed higher values in koji samples with DOM 0–9 substrates, except for

211

samples with DOM 11. Interestingly, amylase activity for all koji types regardless of DOM

212

was highest at 24 h, while protease and β-glucosidase activities increased linearly until 96 h.

213

Measurement of the functional phenotypes, i.e., ABTS and FRAP, to determine antioxidant

214

activities showed that the rice koji samples with DOM 0 substrates had the highest

215

antioxidants levels followed by DOM 5 > DOM 7 >DOM 9, 11 substrates made rice koji

216

(Figure 3 D and E). Hence, we assume that the antioxidant activities of koji increased linearly

217

until 96 h, regardless of DOM of the substrates.

218

4. DISCUSSION

219

We employed the metabolomic approaches to evaluate the comprehensive metabolic and

220

biochemical events underlying fermentative koji preparation with rice substrates having

221

varying DOM. We observed that the differential metabolomes of rice koji types were the

222

direct biochemical functions of (1) time-correlated metabolism, and (2) enzyme activities, by

223

koji mold subjected to fermentation on rice substrates with varying DOM. During the course

224

of fermentative growth, the spore count for A. oryzae was steadily increased while the

225

moisture content was decreased. There were no significant differences observed for mold

226

growth and sporulation among the koji samples with 5 different DOMs. We assumed that the

227

decrease of moisture content in koji was probably due to consumption of water by Aspergillus

228

growth as well as through evaporation.18

229

The multivariate analysis for various rice koji extracts based on GC-TOF-MS datasets

230

indicated a fermentation time-correlated metabolic profile for primary metabolites regardless

231

of the DOM (Figure 1A). On the other hand, the multivariate analysis based on UHPLC11



232

LTQ-ESI-IT-MS/MS datasets showed a secondary metabolite profile depending on various

233

DOM of rice substrates (Figure 1B). As shown in Figure 2, the primary metabolites altered

234

briskly with enzyme activities over time i.e., increased abundance of metabolites related to

235

sugar metabolism with increase in amylase and β-glucosidase activities at 24 h and 72 h,

236

respectively. Similarly, the gradual changes in amino acid levels were recorded with

237

enhanced protease activity. The fatty acid levels were also increased at 24 h.

238

4.1. Time Correlated Metabolic Alterations for Rice Koji Independent of Substrate's

239

DOM. The organic acids produced in the TCA cycle viz., citrate, fumarate, malate, and

240

succinate acts as intermediates to various metabolic pathways (Figure 2). With an increase in

241

organic acids contents, the pH decreased with an accompanying increase in titratable acidity

242

of the rice koji samples (Figure 4C and D). The production of citric acid used in the dairy,

243

food, beverage, pharmaceutical, and biochemical industries is extensively carried out using

244

Aspergillus species.19 The fumaric acid is used as a starting metabolite for the synthesis of

245

polymers, while the malate and succinate has wide applications in the food, beverage, and

246

pharmaceuticals industries.20

247

Among the sugars, the glucose levels showed the highest levels at 24 h unlike other sugar

248

metabolites, followed by its steep decline levels during the later stages of koji fermentation.

249

In particular, glucose is a major carbon source hydrolyzed from the substrate starch by fungal

250

enzymes including amylase and β-glucosidases. The sugars are further metabolized to sugar

251

alcohols through alcoholic fermentation.21 The sugar alcohols are produced during microbial

252

fermentation through multiple fermentation pathways.22 For example, erythritol, a four-

253

carbon sugar alcohol that is widely distributed in nature such as in foods, is produced

254

industrially via the pentose phosphate pathway beginning with enzymatic hydrolysis of the

255

starch from rice to generate glucose. Similarly, Sorbitol can be obtained by simultaneous 12


Page 12 of 33

Page 13 of 33

256


hydrolysis and reduction of glucose to change the aldehyde group to a hydroxyl groups.

257

The proteolytic enzymes produced by Aspergillus species release the organic nitrogen

258

contents from complex proteins to be used as a nitrogen source essential for growth and

259

metabolism.23,24 The rice-koji fermentation with Aspergillus spp. effectively increased the

260

contents of free amino acids (alanine, glycine, and serine) related to sweetness and nutritional

261

properties.25 The total amino acid contents including those of aromatic amino acids

262

(phenylalanine and tryptophan) and branched-chain amino acids (leucine, isoleucine, and

263

valine) were increased with enhanced protease activities over time (Figure 2, 3B, & 4F).

264

Functionally, the biosynthesis of aromatic amino acid in A. fumigatus has been linked with its

265

antifungal properties, while the branched-chain amino acids in A. nidulans are reportedly

266

vital for building proteins.26,27 In our study, we observed a decrease in glutamate contents

267

coupled with an increase in other amino acids contents, especially those of γ–aminobutyric

268

acid (GABA) over time. It has earlier been reported that, A. oryzae contains a gene for

269

glutamate decarboxylase (GAD, E.C. 4.1.1.15) which produces GABA via decarboxylation of

270

glutamic acid during cultivation of the spore suspension.28 GABA was employed in

271

functional foods for reducing blood pressure, promoting better sleep, and diuretic effects.29-31

272

The relative levels of unsaturated fatty acids, saturated fatty acids, and lysophospholipids

273

were increased with the biosynthesis as well as bioconversion of fatty acids (Figure 2).

274

Whole rice bran lipids induce the lipid metabolism. This means that fungi generate excess

275

lipids by fermenting the substrate materials as well as synthesize their own lipids for fungal

276

biomass production.32 Earlier, Abu et al. have reported that solid-state fermentation with A.

277

oryzae typically increases the concentrations of lipids, primarily including C16:0, C18:0, and

278

C18:1.33

279

4.2. Metabolic Alterations for Rice Koji Depending on Substrate's DOM. A rice grain has 13



280

different biomolecular compositions for its different parts arranged from surface to inside

281

including outer husk or hull, bran, embryo, and innermost endosperm. A variety of

282

metabolites in rice viz., phenolic acids, cinnamic acids, anthocyanins, flavonoids, steroidal

283

compounds, polymeric carbohydrate, and proteins are nutritionally vital as health-promoting

284

functional foods.34 Following the milling process, rice contains less of the embryo bud and

285

bran layer (Table 1). We detected higher proportions of amino acids, organic acids, sugars and

286

sugar alcohols, and lysophospholipids together contributing to the high antioxidant activities

287

for DOM 0 rice koji as compared to those for DOM 5–11 koji samples (Table 2, Figure 3 D

288

and E), maintaining similar pH and titratable acidity (Figure 4 C–D). Though, the relative

289

abundance for most metabolites in rice koji, made using the substrates with intermediate

290

DOM, were increased during the course of fermentation, the metabolite abundance for

291

unfermented DOM 0 substrate was comparatively higher than substrates with increased DOM

292

(Table 2).

293

Given the proportion of the bran layer in rice after different DOM, rice koji was relatively

294

affected by various microbial enzymes and associated bioactivities. The protease and β-

295

glucosidase activities were observed varying in the order of substrate's DOM 5 > 7 > 0 > 9 >

296

11, while the amylase activity was observed unaffected by the DOM. Accordingly, amino

297

acids, sugars, and sugar metabolism were highest in DOM 5-7 sample during the middle of

298

fermentation at 24–72 h. Intriguingly, most of the primary metabolites were highest in the

299

koji samples with DOM 0, at the end 96 h fermentation. Mechanistically, β-Glucosidase

300

cleaves glucosidic linkages in polysaccharides and flavonoid glycosides and hydrolyzes them

301

into oligo- or mono-saccharides and corresponding flavonoid aglycones to improve

302

enzymatic saccharification.35 In the koji samples with DOM 5 substrate, enhanced β-

303

glucosidase activities in rice koji were mainly correlated with a decrease in the flavonoid 14


Page 14 of 33

Page 15 of 33


304

glycosides and tricin rutinoside with corresponding increase in the relative levels of sugars

305

and flavonoid aglycone, tricin. The changes observed in the metabolite levels for DOM 0 koji

306

samples might have been impeded by the rice bran wall impervious to enzymatic hydrolysis.

307

Hence, following the rice bran hydrolysis, the relative levels of most metabolites was altered

308

in DOM 0 koji samples. The delay in metabolite release in case of brown rice koji

309

fermentation was reportedly linked to the impenetrable brine layer, which limits enzymatic

310

penetration and subsequent hydrolysis.10 Earlier, Yu et al. have reported that the protease and

311

glucoamylase activities for rice koji made using a brown rice substrate with 50–70% DOM

312

reach maximum following the 5 days fermentation with A. oryzae.36

313

The ABTS and FRAP assay determined antioxidant activities were considerably higher

314

in rice koji using substrates with DOM 0, 5, and 7, in comparison to those for DOM 9 and 11

315

koji samples, during the courses of fermentation. Hence, increasing the substrate's DOM

316

allows the enzymatic penetration through impervious rice bran and endosperm, hence

317

releasing the polyphenols, flavonoids, and free amino acids affecting the antioxidant

318

activities.13,14 Antioxidant activity prevents or limits oxidation, which is considered beneficial

319

for improving food quality and health. Evaluating antioxidant compounds from different

320

DOM of rice substrates provides useful information for commercial koji making owing to the

321

nutritional and functional properties of antioxidant metabolites.

322

Previously, it has been shown that flavonoid and phenol compounds derived from plants

323

possess high antioxidant activities.37 Pérez et al. have reported that the amino acid

324

composition of honey affects its free radical scavenging capacity.38 A known antioxidant

325

phytochemical i.e., ferulic acid is commonly found in rice bran, which reportedly alleviates

326

oxidative stress in various organic systems.39 We observed that the 96 h Rice koji with DOM

327

0 substrate contained the highest levels of antioxidant compounds. We conjecture that 15



328

antioxidant potentials of rice koji are mainly influenced by its flavonoids and phenolic acid

329

components, which synergistically constitutes to its various functional properties.

330

In conclusion, we observed that A. oryzae exhibited a unique overall metabolism

331

maneuvering the secretion of hydrolytic enzymes (amylase, β-glucosidase, and proteases)

332

ergo the metabolomes in rice koji fermented using the substrates with varying DOM.

333

Typically at the initial and final stages of koji fermentation, a variety of functional

334

metabolites were relatively higher in koji made with substrates having DOM 0, in comparison

335

to koji types made with substrates subjected to varying DOM. However, in the middle stages

336

of koji fermentation, the substrates with DOM 5–7 showed relatively higher contents of

337

metabolites intermediates of carbohydrate metabolism viz., sugars and sugar alcohols, organic

338

acids, phenolic acids, as well as lipid metabolism intermediates such as fatty acids and

339

lysophospholipids. The enhanced release of free metabolites in the koji samples was

340

influenced by the relatively higher amylase, β-glucosidase, and protease levels in rice

341

substrates with DOM 5-7 as compared to DOM 0. The present findings provide useful

342

insights for large-scale commercial production of rice koji that relies heavily on the DOM to

343

rationalize its raw substrate processing.

16


Page 16 of 33

Page 17 of 33


344

AUTHOR INFORMATION

345

Corresponding Author

346

*Phone: +82-2-2049-6177; fax: +82-2-455-4291; E-mail: [email protected]

347

Notes

348

The authors declare no competing financial interest.

349 350

SUPPORTING INFORMATION

351

The list of significantly distinct metabolites from rice koji with different DOM during

352

fermentation identified by GC-TOF-MS (Table S1), list of significantly distinct metabolites

353

from rice koji with different DOM during fermentation identified by UHPLC-LTQ-ESI-IT-

354

MS/MS (Table S2), partial least square discriminant analysis (PLS-DA) score plot derived

355

from the GC-TOF-MS (A) and UHPLC-LTQ-ESI-IT-MS/MS data set of rice koji cultured

356

with A. oryzae according to the degree of milling (DOM) (Figure S1).

357 358

REFERENCES

359

1. Ray, R.C.; Montet, D. Microorganisms and fermentation of traditional foods. New York:

360

CRC Press, (Chapter 1), 2014.

361

2. Shin, D.; Jeong, D. Korean traditional fermented soybean products: Jang. J. Ethn. Foods

362

2015, 2, 2-7.

363

3. Bavaresco, L.; Vezzulli, S.; Civardi, S.; Gatti, M.; Battilani, P.; Pietri, A.; Ferrari, F. Effect

364

of Lime-Induced Leaf Chlorosis on Ochratoxin A, trans-Resveratrol, and ε-Viniferin

365

Production in Grapevine (Vitis vinifera L.) Berries Infected by Aspergillus carbonarius. J.

366

Agric. Food Chem. 2008, 56, 2085-2089. 17



367

4. Andújar-Ortiz, I.; Pozo-Bayón, M. A.; García-Ruiz, A.; Moreno-Arribas, M. V. Role of

368

specific components from commercial inactive dry yeast winemaking preparations on the

369

growth of wine lactic acid bacteria. J. Agric. Food Chem. 2010, 58, 8392-8399.

370

5. Chancharoongpong, C.; Hsieh, P.C.; Sheu, S.C. Enzyme production and growth of

371

Aspergillus oryzae S. on soybean koji fermentation. APCBEE Procedia 2012, 2, 57-61.

372

6. Bechman, A.; Phillips, R.D.; Chen, J. Changes in selected physical property and enzyme

373

activity of rice and barley koji during fermentation and storage. J. Food Sci. 2012, 77, 318-

374

322.

375

7. Zhu, Y.; Tramper, J. Koji-where East meets West in fermentation. Biotechnol. Adv. 2013,

376

31, 1448-1457.

377

8. Lee, D.E.; Lee, S.; Jang, E.S.; Shin, H.W.; Moon, B.S.; Lee, C.H. Metabolomic profiles of

378

Aspergillus oryzae and Bacillus amyloliquefaciens during rice koji fermentation. Molecules,

379

2016, 21, 773.

380

9. Kim, A.J.; Choi, J.N.; Kim, J.; Kim, H.Y.; Park, S.B.; Yeo, S.H.; Choi, J.H.; Liu, K.H.; Lee,

381

C.H. Metabolite profiling and bioactivity of rice koji fermented by Aspergillus strains. J.

382

Microbiol. Biotechnol. 2012, 22, 100-106.

383

10. Lee, D.E.; Lee. S.; Singh, D.; Jang, E.S.; Shin, H.W.; Moon, B.S.; Lee, C.H. Time-

384

resolved comparative metabolomes for koji fermentation with brown-, white-, and giant

385

embryo-rice. Food Chem. 2017a, 231, 258-266.

386

11. Yoshizaki, Y.; Yamato, H.; Takamine, K.; Tamaki, H.; Ito, K.; Sameshima, Y. Analysis of

387

volatile compounds in shochu koji, sake koji, and steamed rice by gas chromatography-mass

388

spectrometry. J. Inst. Brew. 2010, 116, 49-55. 18


Page 18 of 33

Page 19 of 33


389

12. Butsat, S.; Siriamornpun, S. Antioxidant capacities and phenolic compounds of the husk,

390

bran and endosperm of Thai rice. Food Chem. 2010, 119, 606-613.

391

13. Liu, L.; Guo, J.; Zhang, R.; Wei, Z.; Deng, Y.; Guo, J.; Zhang, M. Effect of degree of

392

milling on phenolic profiles and cellular antioxidant activity of whole brown rice. Food Chem.

393

2015, 185, 318-325.

394

14. Liu, K.I.; Zheng, J.B.; Chen, F.S. Relationships between degree of milling and loss of

395

Vitamin B, minerals, and change in amino acid composition of brown rice. LWT-Food Sci.

396

Technol. 2017, 82, 429-436.

397

15. Lee, S.; Lee, S.; Singh, D.; Oh, J.Y.; Jeon, E.J.; Ryu, H.S.; Lee, D.W.; Kim, B.S.; Lee,

398

C.H. Comparative evaluation of microbial diversity and metabolite profiles in doenjang, a

399

fermented soybean paste, during the two different industrial manufacturing processes. Food

400

Chem. 2017b, 221, 1578-1586.

401

16. Jang, Y.K.; Shin, G.R.; Jung, E.S.; Lee, S.; Lee, S.; Singh, D.; Jang, E.S.; Shin, D.J.; Kim,

402

H.J.; Shin, H.W.; Moon, B.S.; Lee, C.H. Process specific differential metabolomes for

403

industrial gochujang types (pepper paste) manufactured using white rice, brown rice, and

404

wheat. Food Chem. 2017, 234, 416-242.

405

17. Re, R.; Pellegrini, N.; Proteggente, A.; Pannala, A.; Yang, M.; Rice-Evans, C. Antioxidant

406

activity applying an improved ABTS radical cation decolorization assay. Free Radic. Biol.

407

Med. 1999, 26, 1231-1237.

408

18. Byeon, J.Y.; Choi, E.J.; Kim, W.J. Effect of low frequency (20-35 kHz) airborne

409

ultrasonication on microbiological and physicochemical properties of soybean koji. Food Sci.

410

Biotechnol. 2015, 24, 1035-1040. 19



411

19. Karthikeyan, A.; Sivakumar, N. Citric acid production by koji fermentation using banana

412

peel as a novel substrate. Bioresour. Technol. 2010, 101, 5552-5556.

413

20. Yin, X.; Li, J.; Shin, H.D.; Du, G.; Liu, L.; Chen, J. Metabolic engineering in the

414

biotechnological production of organic acids in the tricarboxylic acid cycle of

415

microorganisms: Advances and prospects. Biotechnol. Adv. 2015, 33, 830-841.

416

21. Kim, A.J.; Choi, J.N.; Kim, J.; Park, S.B.; Yeo, S.H.; Choi, J.H.; Lee, C.H. GC-MS based

417

metabolite profiling of rice koji fermentation by various fungi. Biosci. Biotechnol. Biochem.

418

2010, 74, 2267-2272.

419

22. Godswill, A.C. Sugar alcohols: chemistry, production, health concerns and nutritional

420

importance of mannitol, sorbitol, xylitol, and erythritol. Int. J. Adv. Res. 2017, 3, 31-66.

421

23. Blieva, R.K.; Safuani, Z.E.; Iskakbaeva, Z.A. Effect of various sources of nitrogen and

422

carbon on the biosynthesis of proteolytic enzymes in a culture of Aspergillus awamori 21/96.

423

Appl. Biochem. Microbiol. 2003, 39, 188-191.

424

24. Yousaf, M.; Irfan, M.; ulla Khokhar, Z.; Syed, Q.U.A.; Baig, S.; Iqbal, A. Enhanced

425

production of protease by mutagenized strain of Aspergillus oryzae in solid substrate

426

fermentation of rice bran. Sci. Int. 2010, 22, 119-123.

427

25. Kim, B.M.; Park, J.H.; Kim, D.S.; Kim, Y.M.; Jun, J.Y.; Jeong, I.H.; Nam, S.Y.; Chi, Y.M.

428

Effects of rice koji inoculated with Aspergillus luchuensis on the biochemical and sensory

429

properties of a sailfin sandfish (Arctoscopus japonicas) fish sauce. Int. J. Food Sci. Technol.

430

2016, 51, 1888-1899.

431

26. Sasse, A.; Hamer, S.N.; Amich, J.; Binder, J.; krappmann, S. Mutant characterization and

432

in vivo conditional repression identify aromatic amino acid biosynthesis to be essential for 20


Page 20 of 33

Page 21 of 33


433

Aspergillus fumigatus virulence. Virulence, 2016, 7, 56-62.

434

27. Shimizu, M.; Fujii, T.; Masuo, S.; Takaya, N. Mechanism of De Novo branched-chain

435

amino acid synthesis as an alternative electron sink in hypoxic Aspergillus nidulans cells.

436

Appl. Environ. Microbiol. 2010, 76, 1507-1515.

437

28. Kadir, S.A.; Wan-Mohtar, W.A.A.Q.I.; Mohammad, R.; Lim, S.A.H.; Mohammed, A.S.;

438

Saari, N. Evaluation of commercial soy sauce koji strains of Aspergillus oryzae for γ–

439

aminobutryic acid (GABA) production. J. Ind. Microbiol. Biotechnol. 2016, 43, 1387-1395.

440

29. Hayakawa, K.; Kimura, M.; Kasaha, K.; Matsumoto, K.; Sansawa, H.; Yamori, Y. Effect

441

of a γ-aminobutyric acid enriched dairy product on the blood pressure of spontaneously

442

hypertensive and normotensive Wistar-Kyoto rats. Brit. J. Nutr. 2004, 92, 411-417.

443

30. Yamatsu, A.; Yamashita, Y.; Maru, I.; Yang, J.; Tatsuzaki, J.; Kim, M. The improvement

444

of sleep by oral intake of GABA and Apocynum veneetum leaf extract. J. Nutr. Sci. Vitaminol.

445

2015, 61, 182-187.

446

31. Jakobs, C.; Jaeken, J.; Gibson, K.M. Inherited disorders of GABA metabolism. J. Inher.

447

Metab. Dis. 1993, 16, 704-715.

448

32. Oliveira, M.S.; Feddern, V.; Kupski, L.; Cipolatti, E.P.; Badiale-Furlong, E.; Souza-

449

Soares, L.A. Changes in lipid, fatty acids and phospholipids composition of whole rice bran

450

after solid-state fungal fermentation. Bioresour. Technol. 2011, 102, 8335-8338.

451

33. Abu, O.A.; Tewe, O.O.; Losel, D.M.; Onifade, A.A. Changes in lipid, fatty acids and

452

protein composition of sweet potato (Ipomoea batatas) after solid-state fungal fermentation.

453

Bioresour. Technol. 2000, 72, 189-192. 21



454

34. Friedman, M. Rice brans, rice bran oils, and rice hulls: composition, food and industrial

455

uses, and bioactivities in humans, animals, and cells. J. Agric. Food Chem. 2013, 61, 10626-

456

10641.

457

35. Rani, V.; Mohanram, S.; Tiwari, R.; Nain, L.; Arora, A. Beta-glucosidase: Key enzyme in

458

determining efficiency of cellulose and biomass hydrolysis. J. Bioprocess Biotech. 2014, 5, 1-

459

8.

460

36. Yu, K.W.; Lee, S.E.; Choi, H.S.; Suh, H.J.; Ra, K.S.; Choi, J.W.; Hwang, J.H.

461

Optimization for rice koji preparation using Aspergillus oryzae CJCM-4 isolated from a

462

korean traditional meju. Food Sci. Biotechnol. 2012, 21, 129-135.

463

37. Kong, S.; Lee, J. Antioxidants in milling fractions of black rice cultivars. Food Chem.

464

2010, 120, 278-281

465

38. Pérez, R.A.; Iglesias, M.T.; Pueyo, E.; González, M.; de Lorenzo, C. Amino acid

466

composition and antioxidant capacity of Spanish honeys. J. Agric. Food Chem. 2007, 55,

467

360-365.

468

39. Srinivasan, M.; Sudheer, A.R.; Menon, V.P. Ferulic acid: Therapeutic potential through its

469

antioxidant property. J. Clin. Biochem. Nutr. 2017, 40, 92-100.

470 471

FUNDING SOURCES

472

This work was supported by the National Research Foundation of Korea(NRF) grant funded

473

by the Korea government(MSIP) (No. NRF-2017M3C1B5019303) and funded by the

474

Strategic Initiative for Microbiomes in Agriculture and Food, Ministry of Agriculture, Food

475

and Rural Affairs, Republic of Korea (as part of the (multi-ministerial) Genome Technology 22


Page 22 of 33

Page 23 of 33

476


to Business Translation Program) (grant number 916005-2).

23



477

FIGURE CAPTIONS

478

Figure 1. Principal component analysis (PCA) score plot from the GC-TOF-MS (A) and

479

UHPLC-LTQ-IT-MS/MS (B) data set of rice koji cultured with A. oryzae according to the

480

degree of milling (DOM) ) (◆, ◆, 0; ◆, 5; ◆, 7; ◆, 9; ◆, 11 ; +, 0 hr; ✱, 12 h; ▲, 24 h; ■,

481

36 h; ▼, 48 h; ●, 72 h; ◆, 96 h).

482 483

Figure 2. Pathway for the relative levels of discriminant metabolites at different times of rice

484

koji with DOM as determined using the PLS-DA datasets (variable importance in projection

485

> 1.0, p-value < 0.1) for GC-TOF-MS analyses. The discriminant metabolites were further

486

correlated with corresponding steps in the biosynthetic pathways adapted from KEGG

487

database. The values represent the fold-change with respect to unfermented rice.

488 489

Figure 3. Changes in amylase (A), protease (B), β-glucosidase (C), ABTS (D), and FRAP (E)

490

of rice koji cultured with A. oryzae according to DOM during fermentation (■, 0; ■, 5; ■, 7;

491

■, 9; ■, 11).

492 493

Figure 4. Total mold count (A), moisture contents (B), pH (C), titratable acidity (D), total

494

sugar content (E), and total amino acid content (F) of koji cultured with A. oryzae according

495

to the degree of milling (DOM) during fermentation (■, 0; ■, 5; ■, 7; ■, 9; ■, 11).

24


Page 24 of 33

Page 25 of 33


496 497

Figure 1.

498

25



499 500

Figure 2.

501

26


Page 26 of 33

Page 27 of 33


502 503

Figure 3.

504

27



505 506

Figure 4.

507

28


Page 28 of 33

Page 29 of 33

508


Table 1. Information for degree of milling of rice .

Degree of milling

Proportion of the embryo bud and

(DOM)

bran layer in rice (%)

0

100

5

50

7

30

9

15

11

5

29


509


Page 30 of 33

510

Table 2. Column representations for the highest levels of discriminant metabolites in 5 rice

511

koji according to the DOM. Each column represents DOM color having metabolite of the

512

highest content at each time. (■, 0; ■, 5; ■, 7; ■, 9; ■, 11)

Fermentation time Metabolites

0h 7

12 h

0

5

9

11

0

alanine

'

'

'

'

'

asparagine

'

'

'

'

aspartic acid

'

'

'

γ-Aminobutyric acid

'

'

glutamic acid

'

glycine

24 h

5

7

9

11

0

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

Isoleucine

'

'

'

'

leucine

'

'

'

lysine

'

'

methionine

'

orinithine

36 h

5

7

9

11

0

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

phenylalanine

'

'

'

'

'

'

proline

'

'

'

'

'

pyroglutamic acid

'

'

'

'

serine

'

'

'

threonine

'

'

tryptophan

'

valine

48 h

5

7

9

11

0

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

myristic acid

'

'

elaidic acid

'

linoleic acid

72 h

5

7

9

11

0

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

linolenic acid

'

'

'

'

oleic acid

'

'

'

palmitic acid

'

'

stearic acid

'

'

96 h

5

7

9

11

0

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

5

7

9

11

'

'

' '

'

'

'

' '

'

'

'

'

' '

'

'

'

'

'

' '

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

Amino acids

Fatty acids

Organic acids lactic acid

30


Page 31 of 33


4-hydroxybenzoic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

acetic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

citric acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

ethanolamine

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

fumaric acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

malic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

salicylic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

shikimic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

succinic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

ferulic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

glycerol

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

adonitol

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

dulcitol

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

gluconic acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

Sugar & sugar alcohols

glucose glyceric acid

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

glyceryl-glycoside

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

maltose Erythritol

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

myo-inositol

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

N-acetyl-D-glucosamine

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

raffinose

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

sorbitol

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

sucrose

'

'

'

'

'

'

'

'

' '

xylose

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

adenine

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

guanine

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

olemide

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

uridine

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

'

' '

Nucleosides

Secondary metabolites apigenin-6-C-glucosyl-8-C-arabinoside isovitexin-2''-O-glucoside tricin-7-O-rutinoside tricin pinellic acid lysoPE14:0

31



lysoPC18:3 lysoPC14:0 lysoPE18:2 lysoPC16:1 lysoPC18:2 lysoPE16:0 lysoPC18:1

513 514

32


Page 32 of 33

Page 33 of 33

515


GRAPHIC FOR TABLE OF CONTENTS

516 517 518 519

33


Metabolomics Reveal Optimal Grain ... - ACS Publications

Recommend Documents