Mass Spectrometry-Based ... - ACS Publications

Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA

Omics Technologies Applied to Agriculture and Food

GC/MS-based Metabolites Profiling of Nutrients and Antinutrients in Eight Lens and Lupinus Seeds (Fabaceae) Mohamed A. Farag, Amira R. Khattab, Anja Ehrlich, Matthias Kropf, Andreas G. Heiss, and Ludger A. Wessjohann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00369 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 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.

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 42

Journal of Agricultural and Food Chemistry

1

For submission to: JAFC

2

GC/MS-based Metabolites Profiling of Nutrients and Anti-

3

nutrients in Eight Lens and Lupinus Seeds (Fabaceae)

4

Mohamed A. Farag1,2*, Amira R. Khattab3, Anja Ehrlich4, Matthias Kropf5, Andreas G. Heiss6,

5

Ludger A. Wessjohann4

6

1

Pharmacognosy Department, College of Pharmacy, Cairo University, Cairo, Egypt, Kasr el Aini St., P.B. 11562;

7 2

8

Chemistry Department, School of Sciences & Engineering, The American University in Cairo, New Cairo 11835, Egypt;

9 3

10

Academy for Science, Technology and Maritime Transport, Alexandria, Egypt, P.B.1029;

11 4

12

Institute for Integrative Nature Conservation Research, University of Natural Resources and Life Sciences Vienna (BOKU), Austria;

13 14

5

Institute for Integrative Nature Conservation Research, University of Natural Resources and Life Sciences Vienna (BOKU), Austria;

15 16 17

Pharmacognosy Department, Division of Pharmaceutical Sciences, College of Pharmacy, Arab

6

Department for Bioarchaeology, Austrian Archaeological Institute (ÖAI), Austrian Academy of Sciences (ÖAW), Austria.

18 19

*Corresponding author: Mohamed A. Farag

20

E-mail: [email protected], [email protected]

21

Tel: +011-202-2362245, Fax: +011-202-25320005

22 23 24

ACS Paragon Plus Environment


25

ABSTRACT

26

Lens culinaris and several Lupinus species are two legumes regarded as potential protein

27

resources aside from their richness in phytochemicals. Consequently, characterization of their

28

metabolite composition seems warranted to be considered as a sustainable commercial

29

functional food. This study presents a discriminatory-holistic approach for metabolites

30

profiling in accessions of four lentil cultivars and four lupinus species via gas

31

chromatography-mass spectrometry. A total of 107 metabolites were identified encompassing

32

organic and amino acids, sugars and sterols along with anti-nutrients viz. alkaloids and sugar

33

phosphates. Among the examined specimens, four nutritionally-valuable accessions ought to

34

be prioritized for future breeding to include Lupinus hispanicus, enriched in organic

35

(ca.11.7%) and amino acids (ca.5%), and L. angustifolius, rich in sucrose (ca.40%), along

36

with two dark-colored lentil cultivars ‘verte du Puy’ and ‘Black Beluga’ enriched in peptides.

37

Anti-nutrient chemicals were observed in Lupinus polyphyllus owing to its high alkaloids

38

content. Several species-specific markers were also revealed using multivariate data analyses.

39 40

KEYWORDS: Functional foods; GC/MS; Lens culinaris; Lupinus; Metabolite profiling;

41

Nutrients; OPLS-DA; PCA.

42

43

44

45

46


Page 2 of 42

Page 3 of 42


47

1. Introduction

48

Increasing demand for protein resources owing to the growing human population along

49

with the reported health risk of animal protein consumption warrants the development of

50

other plant protein as in soybean derived food. Such interest towards the incorporation of

51

plant proteins in human diet derived the development of effective analytical methods for the

52

screening of plant foods.1

53

Leguminous seeds, viz. lupines, soybeans, lentils, beans, and peas are valued as

54

promising alternative supply of proteins for human and animal consumption worldwide.

55

Among these, lupin (Lupinus L.) is one of the richest protein resources, almost as high as that

56

of soybean (~35-40% of the dry weight) followed by lentil (Lens culinaris Medik.), which is

57

known as “poor man’s meat”, with a protein content of ca. 20–25%. It should be noted that

58

compared to animal protein, plants present a less complete resource of amino acids which

59

warrants mixing different plant protein diet in what is known typically as complementary

60

protein. 2-3

61

Both Lens culinaris and Lupinus seeds provide a well-balanced source of essential amino

62

acids, carbohydrates, fiber, minerals, and vitamins as well as a myriad of bioactive

63

phytochemicals viz., quinolizidine alkaloids, polyphenols and saponins of value for the

64

management and prevention of ailments.4 However, these seeds contain a number of anti-

65

nutrients, i.e. tannins, oligosaccharides, saponins, phytates, and protease inhibitors, well

66

reported to cause detrimental effects to human nutritional status by hindering the uptake and

67

utilization of minerals, vitamins, proteins and other key nutrients. 3

68

Lupinus species (subfamily Faboideae; tribus Genisteae), such as L. albus (white lupin),

69

L. angustifolius (blue lupin), L. luteus (yellow lupin), L. mutabilis (pearl lupin) and L.

70

hispanicus (Spanish lupin), have been domesticated owing to their nutritive value and ease of



Page 4 of 42

71

cultivation under different climatic conditions.1 These species are widely distributed and,

72

according to Gladstones (1998) are grouped into “Old World Species”, located in the

73

Mediterranean-African region, while “New World Species” include the species found in

74

North and South America such as L. polyphyllus. For the three “Old World Species” under

75

study (i.e. Lupinus angustifolius, L. hispanicus, and L. luteus; Table 1) a phylogenetic close

76

relationship has been demonstrated (cf. Käss & Wink 1997).5 Overall, lupines are

77

characterized by a diverse geographical distribution, morphological variation and

78

chromosomal polymorphisms.6

79

The health benefits of lupin have been extensively reported to include hypoglycemic,

80

hypotensive, cholesterol lowering, anticancer and anti-inflammatory effects aside from their

81

protection against menopausal symptoms and osteoporosis.1, 7

82

All Lupinus species produce quinolizidine alkaloids such as lupinine, lupanine and α-

83

isolupanine, albeit with qualitative and quantitative differences as revealed

84

multivariate statistical tools.8 The phenolic secondary metabolites in Mediterranean lupin

85

species have been reported mostly using LC/MS8-10, particularly considering their value as

86

chemotaxonomic markers of Lupinus spp. than the classically employed morphological and

87

cytological features.11 Being also a member of the Fabaceae family (subfamily Faboideae),

88

lentil seeds (tribus Fabeae) possess a comparable bioactive composition to lupin with

89

biological effects to include antioxidant, anticarcinogenic, antihyperglycemic and

90

hypocholestrolemic. Lentil is classified into several market classes i.e., extra small red, small

91

red, large red, small green, medium green, large green, Spanish brown, zero tannin and Puy.12

92

The profiling of phenolic secondary metabolites of the various lentil genotypes was

93

reported in relation to its antioxidant capacity.13-14 Phenotypic selection of lentils with


using

Page 5 of 42


94

improved nutritional attributes was further achieved based on the selection of a wide range of

95

molecular markers.15

96

With an increasing interest in a rather holistic view for metabolites composition in

97

legumes, the development of large scale analytical methods now follows. Plant metabolomics

98

is valued as a comprehensive profiling technology employed to obtain snapshots of all low

99

molecular weight molecules (the metabolome) in a given organism. Such approach

100

commonly utilizes rigorous hyphenated mass spectrometry techniques such as gas or liquid

101

chromatography mass spectrometry (GC/MS) and (LC/MS), applied in food and

102

nutraceuticals analysis. The complexity and richness of metabolomics data can only be

103

harnessed using a diverse set of chemometric tools, such as classification, dimensionality

104

reduction, visualization, pattern recognition, and or modelling.16 Such platforms can be

105

employed to confirm the identity of taxonomically-related plant species, monitor its quality

106

or purity attributes and/or to standardize plant-derived products.17-19 The untargeted GC/MS-

107

based metabolomics is routinely used to record the rich matrix of plants, made up of several

108

low molecular weight metabolites.

109

There were scarce reports for multivariate data analyses application for the classification

110

of examined seeds, except for the application of artificial neural network and PCA for the

111

discrimination of two Lupinus species, L. albus and angustifolius.

112

the current study was to profile Lens and Lupinus seed accessions metabolites to provide

113

better insight into their compositional differences and/or nutritional traits. Considering the

114

complexity of acquired data, unsupervised and supervised multivariate data analyses viz.

115

principal component analysis (PCA) and orthogonal partial least squares (OPLS), were

116

employed for classification of seed samples, and to ensure good analytical rigorousness.

117

2. MATERIALS AND METHODS


20

The main objective of


118

2.1. Plant material

119

Lens culinaris and Lupinus seeds were obtained from two main sources: seed exchange

120

with four Botanical Gardens (BAS, HOH, IB, COI), as well as two Austrian organic food

121

vendors. Details are listed in Table 1.

122

2.2. Chemicals

123

All solvents used were of LC/MS grade purchased from J. T. Baker (The Netherlands).

124

Umbelliferone was procured from ChromaDex (LGC Standards, Wesel, Germany). All other

125

chemicals and standards were purchased from Sigma Aldrich (St. Louis, MO, USA).

126

2.3. Sample preparation for GC/MS analysis

127

Dried seeds were ground using pestle and mortar under liquid nitrogen. The obtained

128

powder (30 mg) was then homogenized with 2.5 mL 100% MeOH containing 5 µg/mL

129

umbelliferone (an internal standard for relative quantification) using a Turrax mixer operated

130

at 11.000 rpm for 20 s for 5 periods, with 1 min of recession between each mixing period to

131

guard against excess heat generated during mixing. For the removal of plant debris, the

132

extracts were vortexed vigorously and centrifuged at 3000 g for 30 min.

133

2.4. GC/MS analysis of silylated primary metabolites

134

For analysis of primary metabolites (viz. amino acids, organic acids, and sugars), 100 µL

135

of extract (prepared as described in section 2.3) was evaporated to dryness under nitrogen.

136

Derivatization with 150 µL of N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA)

137

was performed at 60◦C for 45 min. Samples were equilibrated at 28◦C prior to analysis using

138

Shimadzu GC-17A gas chromatograph interfaced with Shimadzu QP5050A mass

139

chromatograph. Separation of silylated derivatives was carried out on Rtx-5MS (30 m length,

140

0.25 mm inner diameter, and 0.25 µm film) column. Injections were made in the split mode

141

with a split ratio of 1:15 under the following conditions: injector 280˚C, column oven 80˚C


Page 6 of 42

Page 7 of 42


142

for 2 min, then programmed at a rate of 5˚C/min to 315˚C, kept at 315˚C for 12 min. He

143

carrier gas at 1 mL min-1. The transfer line and ion–source temperatures were adjusted at

144

280°C and 180˚C, respectively. The HP quadrupole mass spectrometer was operated in the

145

electron ionization mode (EI, 70 eV), with a scan range of 50-650 m/z. The identification of

146

the silylated compounds was performed according to the procedure described in 21 and peaks

147

were first deconvoluted using AMDIS software (www.amdis.net) and identified by its

148

retention indices (RI) relative to n-alkanes (C8-C40), mass spectrum matching to NIST,

149

WILEY library database and with authentic standards whenever available.

150

2.5. Multivariate data analysis

151

Principal component analysis (PCA) and orthogonal partial least squares-discriminate

152

analysis (OPLS-DA) were performed with the program SIMCA-P Version 13.0 (Umetrics,

153

Umeå, Sweden). The PCA was run for obtaining a general overview of the variance of

154

metabolites in the different seed accessions under study, and for obtaining information on

155

differences in the metabolite composition among species was revealed by OPLS-DA.

156

Performance of chemometric models was evaluated using the two parameters Q2 and R2,

157

where R2 is employed to quantify the goodness-of-fit of the model; whereas the model

158

predictability is determined from Q2 values. The distance to the model (DModX) was used

159

for detecting outliers. Iterative permutation test was further done to omit the non-randomness

160

of separation between groups.

161

2.6. Statistical Analysis

162

Statistical analyses were performed by SPSS (Statistical Package for Social Science) for

163

Windows software, version 23.0 (SPSS Inc., Chicago, IL, USA). One-way ANOVA followed

164

by post hoc Tukey's test was used at significance level less than 0.05 for multiple



165

comparisons between mean values of the seed metabolites from three biological replicates to

166

determine significant differences among the examined seed accessions.

167

3. RESULTS AND DISCUSSION

168

The nutritive value of legume seeds depends on its nutrient as well anti-nutrient

169

chemicals composition. Seeds richness in a wide range of macronutrients viz. proteins,

170

sugars, fatty acids, is rather limited by their anti-nutritive content viz. alkaloids and phytic

171

acid that might have a negative impact on the digestibility and bioavailability of other

172

nutrient elements. Development of analytical methods for measuring both classes is thus

173

warranted in legume seeds. In the current study, two legumes seeds “Lens & Lupinus” were

174

subjected to metabolite profiling using GC/MS targeting its primary and secondary low

175

molecular weight chemicals.

176

3.1.

GC/MS peaks identification in Lens & Lupinus seeds

177

GC/MS analysis was employed for profiling primary metabolites viz. sugars, organic and

178

amino acids, phosphorylated compounds, fatty acids along with low molecular weight or non-

179

polar secondary metabolites exemplified in alkaloids and steroids. The biological variance

180

within each seed accession was assessed by analyzing three independent biological replicates

181

analysed under the same conditions.

182

Chromatograms (Fig. S1) display a representative profile of Lens and Lupinus seed

183

accession metabolome. A total of 107 silylated metabolites were detected belonging to

184

different metabolite classes including sugars, alkaloids, steroids, and amino as well as fatty

185

acids (Table 2).

186

ANOVA analysis revealed significant differences in the percentile levels of phosphate, fatty

187

acids, viz. linoleic, oleic and stearic acids, and sugars, viz. α-D-glucopyranoside and


Page 8 of 42

Page 9 of 42


188

glucopyranose 4-O-β-D-galactopyranosyl, among the different seed accessions with a

189

confidence level of 95%. Lupinus hispanicus Boiss. & Reutt. showed a significantly higher

190

lupinine content than other seed accessions, whereas, α-isolupanine and its 13-hydroxy

191

derivative were significantly higher in L. polyphyllus Lindl. ‘Russell’ at p < 0.05. Differences

192

in organic acids composition viz. succinic acid and its methyl derivative, carbamic and

193

acetic acids, nitrogenous compounds viz. ethanolamine, hydroxylamine, phenylethanolamine,

194

α-Aminoisobutyric acid and cadaverine

195

Medik. subsp. culinaris ‘Petite Rouge d’Egypte’compared to other accessions.”

196

were statistically significant in Lens culinaris

3.1.1. Nutritients in Lens and Lupinus seeds

197

Sugars (mono- and disaccharides) amounted for the major metabolite class in all

198

examined Lens and Lupinus seeds (Table 2), making up to ca.75 % of the total metabolite

199

content except for the two Lupinus species, namely L. hispanicus Boiss. & Reutt. and L.

200

polyphyllus Lindl. ‘Russell’ totaling ca. 48%. A bar chart depicting the major metabolite

201

class percentile levels in Lens and Lupinus seed accessions are represented in Fig. 1,

202

revealing seeds enrichment in sugars followed by organic acids.

203

3.1.1.1. Sugars and sugar alcohols

204

Disaccharides presented the most abundant sugar subclass, with sucrose (G95)

205

amounting for ca.30% of the total monitored peaks except for Lens culinaris Medik. subsp.

206

culinaris ‘Petite Rouge d’Egypte’, with trace sucrose levels, being instead enriched in

207

melibiose (G103, ca.46%) followed by Lupinus luteus L. (ca.18%). Melibiose is formed in

208

legume seeds via an invertase-mediated hydrolysis of the oligosaccharide raffinose with the

209

production of fructose. Raffinose family oligosaccharides “α-galactosyl derivatives of

210

sucrose” are ubiquitous seeds metabolites reported to accumulate during seed development

211

and disappear rapidly post germination concurrent with an increase in sucrose levels.22 Such



Page 10 of 42

212

correlation between sucrose and raffinose was also observed in our seed specimens.

213

Melibiose health benefits include enhanced minerals absorption,

214

microbiota via promoting the growth of beneficial flora, particularly Bifidobacterium

215

and Lactobacillus strains while inhibiting the growth of pathogenic bacteria aside from its

216

immunostimulant and anti-allergic effect..23-25

modulation of gut

217

Sucrose, a dimer of glucose and fructose, was not reported to promote glucose-

218

intolerance; hence, its intake from sucrose-enriched foods in moderate amounts is favored

219

due to the evidenced potentiation of insulin release by fructose that occur in the presence of a

220

stimulatory glucose level.26 Accordingly, the consumption of sucrose rich seed accession

221

under study “L. angustifolius L. Boltensia” is unlikely to cause increase in post prandial

222

glucose levels.27

223

It should be noted that although sugar alcohols were represented by eleven peaks viz. L-

224

Threitol (G74), Meso-Erythritol (G75), Xylitol (G77), D-Arabitol (G78), D-Pinitol (G81), D-

225

Sorbitol (G82 & G83) and Galactinol (G97, G99, G102 & G104), they were present at trace

226

levels comparable to cyclic sugars, and being most abundant in Lens culinaris Medik. subsp.

227

culinaris ‘Petite Rouge d’Egypte’ and Lupinus luteus L.

228

Lens plants were found to minimize sugar alcohol levels acting as humectant upon

229

exposure to high moisture so as to guard against water saturation and decomposition of

230

mature seeds.28

231

Nevertheless, these metabolites inhibit pathogens growth in the gastrointestinal tract and

232

promote the growth of the probiotic strains that possess many health-promoting and

233

immunomodulatory properties.29


Page 11 of 42


234

Galactosyl cyclitols (galactinol) and cyclitols (D-pinitol) are found to accumulate in the

235

legume seeds, functioning as reserve carbohydrates during germination, and for seeds

236

viability.30 In that regard, Lens culinaris Medik. subsp. culinaris ‘Petite Rouge d’Egypte’

237

encompassed the highest galactinol and pinitol levels (16%), followed by Lupinus luteus L.

238

(10%).

239

3.1.1.2. Organic and inorganic acids

240

Organic acids, the odor-imparting compounds to lupin flour, constituted the second most

241

abundant class comprising (5-14%) of the total identified metabolites. Citric acid (G19) is the

242

major identified organic acid (4-7%) followed by malic (G13, 1-3%) and malonic acids (G5,

243

0.1-2%). Among examined seed specimens, Lupinus hispanicus Boiss. & Reutt. and L.

244

polyphyllus Lindl. ‘Russell’ were most enriched in organic acids (ca.12-14%). These acids

245

are most probably produced as a result of amino acids degradation caused by microorganisms

246

present on the seed hulls.29

247

Citric acid exhibits antimicrobial properties due to its acidulation properties in addition

248

to antioxidant effect via metal ions chelation, whereas malic acid is commonly found in

249

unripe fruits and contributes to its sour taste.31-32 It should be noted that the health hazard

250

organic acids viz. oxalic acid was not detected in any of the examined seed extracts.33

251

Phosphoric acid (G55) was found most abundant in Lupinus hispanicus Boiss. & Reutt.

252

(9.7%) and Lens culinaris Medik. subsp. culinaris ‘Black Beluga’ (5.8%) likely derived from

253

phospholipids degradation reported to actively function under high seed moisture content as

254

observed in stored soybean seeds.34 Phosphoric acid can synergize the antioxidant potential

255

of phenolic antioxidants such as flavonoids in legume seeds.32, 34 Inorganic phosphate was

256

also detected in peak (G56), found more abundant in “Old World lupine Species” with the

257

highest level in Lupinus hispanicus Boiss. & Reutt. (11%). Phosphate is an essential nutrient



258

required for vital biological reactions that maintain cell homeostatic aside from serving as a

259

component of genetic material, that is DNA.35

260

3.1.1.3. Amino acids/peptides

261

Examined specimens of both genera were found to be enriched in free essential amino

262

acids, i.e., valine, lysine, threonine, and phenylalanine needed for protein synthesis, growth

263

and maintenance of whole-body homeostasis. Nevertheless, examined specimens all lacked

264

sulfur-containing essential amino acids in accordance with previous reports.36 Compared to

265

lentil amino acid profile, lupine seeds appeared to be more enriched, with L. hispanicus

266

Boiss. & Reutt. encompassing the highest levels at ca. 5% of all detected metabolites.

267

Consequently, Lupinus species can be regarded as a better nutritive source of plant-based

268

protein compared to lens based on free amino acids analysis. Such conclusion needs be

269

however further verified by comparing its crude protein content using proteomic tools.

270

Free amino acids are reported to exist at small levels in non-germinated Lens seeds, with

271

alanine, glutamic, asparagine and aspartic acids the major protein amino acids and in

272

agreement with our results.37 γ-Aminobutyric acid (GABA), previously reported to be found

273

in germinated Lens culinaris seeds at a concentration of 1.64mg/g dry weight37 and to exhibit

274

hypertensive activity38, was detected in all examined seed accessions except L. culinaris

275

‘Medik. subsp. culinaris ‘Petite Rouge d’Egypte’.

276

Legume seeds are reported to contain peptides with higher antioxidant capacity than their

277

intact proteins.39 Two Lens accessions, namely L. culinaris Medik. subsp. culinaris ‘verte du

278

Puy’ (brown colored) and L. culinaris Medik. subsp. culinaris ‘Black Beluga’ were found to

279

contain the highest peptide levels exemplified in Cys-Gly (G68), Glycyl-l-glutamic acid

280

(G69), Ser-Leu (G70) and Ala-Thr (G71).


Page 12 of 42

Page 13 of 42

281


3.1.1.4. Sterols/triterpenes

282

The two dark colored Lens culinaris seed accessions, namely L. culinaris Medik. subsp.

283

culinaris ‘verte du Puy’ (brown colored) and L. culinaris Medik. subsp. culinaris ‘Black

284

Beluga’ were found to be the most abundant in the triterpene i.e., α-amyrin (G73), with

285

reported anti-inflammatory activity40, present at 6 and 3%, respectively. β-sitosterol (G72),

286

major phytosterol in legume seeds41, was detected though at trace levels (0.1%) in most of

287

accessions except Lupinus polyphyllus Lindl. ‘Russell’ and Lens culinaris Medik. subsp.

288

culinaris ‘Petite Rouge d’Egypte’. β-sitosterol present in lentils is likely to mediate in part for

289

its hypocholesterolaemic effect.41

290 291

3.1.2. Anti-nutrient phytochemicals in Lens and Lupinus seeds 3.1.2.1.

Phytic acid

292

Phytic acid “myo-inositol-1, 2, 3, 4, 5, 6 hexabisphosphates”, represented by peaks G86

293

& G91, was the major detected source of phosphorous in seeds, likely to provide a source of

294

inorganic phosphorus during seed development42 and was interestingly found most abundant

295

in the three phylogenetically closely related Old World lupine Species “Lupinus angustifolius

296

L. ‘Boltensia’, L. luteus L. and L. hispanicus Boiss. & Reutt. with the latter species

297

containing the highest levels (ca. 3%). In contrast, only Lens culinaris accession with yellow

298

colored seeds (sample code LCY, Table 1) was found to be enriched in phytic acid at 3%

299

among Lens specimens.

300

Phytic acid is regarded as anti-nutrient forming chelates with mineral and protein and

301

thereby decreasing their bioavailability. Albeit, recent research suggests that phytic acid and

302

other inositol phosphates provide health benefits such as amelioration of heart diseases, and

303

reduced risk of colon cancer.43 Variation in phytic acid levels exists among seed genotype in

304

addition to cultivation i.e., climate and type of soil.43



305

3.1.2.2. Alkaloids/nitrogenous compounds

306

Lupin seeds are generally classified as either bitter or sweet based on their levels of toxic

307

quinolizidine alkaloids.8 From a nutritional point of view, the alkaloid profile is crucial as

308

significant adverse effects could result such as protein indigestibility and neurological

309

disorders.44

310

Two bitter Lupinus seeds were identified in this study mainly L. polyphyllus Lindl.

311

‘Russell’ with highest alkaloid levels i.e., 13-hydroxy-lupanine (11%) and α-isolupanin (8%),

312

followed by L. hispanicus Boiss. & Reutt. encompassing lupinine at ca. 14%. In contrast, L.

313

angustifolius L. ‘Boltensia’ is regarded based on GC/MS results as a sweet lupin as it

314

contains alkaloids at trace levels (0.2%). Interestingly, cadaverine a toxic diamine produced

315

by the decarboxylation of lysine, and serving as precursor for quinolizidine alkaloids,45 was

316

detected at its highest levels in Lens culinaris Medik. subsp. culinaris ‘Petite Rouge

317

d’Egypte’, though with no downstream effect on the accumulation of lupanine alkaloids.

318

Metabolic fate of cadaverine in Lens culinaris has yet to be revealed or its physiological role

319

in its seeds.

320

Eleven other nitrogenous compounds were detected in examined seed accessions

321

amounting for 1-12% of the total monitored peaks. The major identified nitrogenous

322

compound was ethanolamine, accounting for ca. 2.3% in the ‘Petite Rouge d’Egypte’ cultivar

323

of Lens culinaris. Ethanolamines are derived from N-acylethanolamines, plant lipids with

324

anti-inflammatory activity.46 Interestingly, another nitrogenous compound with potential anti-

325

inflammatory activity detected include indole-3-acetamide, reported to inhibit phospholipase

326

A2, was detected at considerable level (9%) in Lupinus hispanicus.47

327

3.2. GC/MS- based multivariate data analysis of Lens & Lupinus seeds


Page 14 of 42

Page 15 of 42


328

Untargeted GC/MS metabolite profiling was further performed in order to unveil the

329

relative metabolites variation embedded within different Lens and Lupinus seed accessions

330

employing multivariate data analyses tools viz. hierarchical cluster analysis (HCA), principal

331

component analysis (PCA) and orthogonal projection to latent structures-discriminant

332

analysis (OPLS-DA). The un-decoded similarities and variabilities among specimens were

333

revealed first via (HCA) using the Ward's algorithm and evaluated based on the distances

334

between the clusters (the squared Euclidean distance). The reproducibility of the extraction

335

and analysis conditions were clearly evident from the tight clustering of the independent

336

biological triplicates within same seed accession as seen in Fig. 2-A.

337

HCA-driven dendrogram (Fig. 2-A) portrayed two main clusters; cluster “1a”, composed

338

of two species which are the sweet Lupinus angustifolius L. ‘Boltensia’ (LU_A) and Lens.

339

culinaris Medik. subsp. culinaris ‘Petite Rouge d’Egypte’ and cluster “1b” in which the rest

340

of the seed accessions was grouped altogether. The sample grouping in 1a cluster could be

341

ascribed to their diminished alkaloidal content concurrent with an enriched sugar profile.

342

Asides, the three lentil accessions LCBK, LCB, and LCY (Table 1) were distinctly clustered

343

in one sub-branch in cluster “1b” signifying their quite compositional resemblance. It should

344

be noted that overall HCA analysis failed to provide clear discrimination between the

345

specimens of the two genera Lens and Lupinus.

346

PCA model, another unsupervised ordination method but with different graphical

347

representation, was generated using same data matrix for discrimination between the different

348

seed accessions. The established model (Fig. 2-B & C) resulted in the formation of two

349

orthogonal PCs, which accounted for 53% of the total variance using only the first two

350

components, i.e., PC1, accounted for 36% of the variance versus 17% for PC2. The PC1/PC2

351

score plot (Fig. 2-B) shows that triplicates of Lens culinaris Medik subsp. culinaris ‘Petite



352

Rouge d’Egypte’ and one of the three replicates of Lupinus hispanicus Boiss. & Reutt. were

353

positioned on the far right side of the plot (positive PC1 values), whereas on the far left side,

354

most of specimens namely “Lens culinaris Medik. subsp. culinaris ‘verte du Puy’ (brown

355

colored), L. culinaris Medik. subsp. culinaris (yellow colored), L. culinaris Medik. subsp.

356

culinaris ‘Black Beluga’ and Lupinus angustifolius” were located (negative PC1 values). The

357

separation observed in PCA can be explained in terms of the annotated metabolites, using the

358

loading plot (Fig 2-C). MS signals for melibiose, having a positive effect on PC1, contributed

359

the most to the discrimination between examined seed accessions which appeared to be more

360

enriched in the LCO lentil samples, Table 1. The separation of Lupinus hispanicus Boiss. &

361

Reutt. samples (Fig. 2-B) along PC2 can be explained in terms of its enrichment in 1H-

362

indole-3-acetamide and lupinine alkaloid, contributing (negatively) to PC2. The segregation

363

observed in the score plot can mostly be ascribed for seeds enrichment in sugars and

364

alkaloids. However, it should be noted that this PCA model was still not efficient in neither

365

capturing the maximal variability embedded within the studied specimens nor discriminating

366

between Lens and Lupinus samples at the genus level.

367

Supervised orthogonal projection to latent structures discriminant analysis (OPLS-DA)

368

was thus employed by modelling all seed accession replicates in one group to build a

369

classification model for achieving better (species) separation. OPLS-DA also has a greater

370

potential in the identification of markers by providing the most relevant variables for the

371

differentiation between two sample groups. The developed model (Fig. S2) showed a better

372

samples separation, though with low R2 (0.279) and Q2 (0.063) values indicating the weak

373

prediction power of this model.


Page 16 of 42

Page 17 of 42


374

OPLS-DA score plot (Fig S2-A) revealed that Lens accession LCO (Table 1) are

375

clustered in one distinct group. Whereas, other three Lens accessions were closely grouped on

376

the left side. Such scattering is attributable to their enrichment in α-D-glucose and sucrose as

377

depicted from their loading plot (Fig. S2-B), and is in agreement with HCA results (Fig. 2-

378

A). The OPLS model suggests that melibiose enrichment can be regarded as a marker for

379

discriminating LCO from the other lentil accessions. Another improvement in samples

380

classification was observed in the dendrogram derived from OPLS analysis (Fig. S2-C),

381

being successful in grouping all the Lupinus species in one cluster “1c”, that is separately

382

from Lens specimens.

383

Another OPLS-DA modelling was performed in which the Lens and Lupinus species

384

were modelled against each other and with the derived score plot showing a clear separation

385

between both samples this time with optimal prediction parameters “R2 (0.9149) and Q2

386

(0.794)”. The derived score plot (Fig. 3-A) depicted a clear discrimination between both

387

species.

388

The observed sample segregation was ascribed to sugars viz. α-D-glucose, D-

389

Glucopyranose, 4-O-[β-D-galactopyranosyl] and α-amyrin enrichment in Lens versus the

390

abundance of citric acid and lupinine alkaloid in Lupinus specimens as revealed from the

391

corresponding S-plot (Fig. 3-B). The alkaloid lupinine can be regarded as a chemotaxonomic

392

marker for Lupinus being almost absent in Lens specimens examined. The positive

393

correlation of citric acid and lupinine in Lupinus accessions suggest that it functions to

394

solubilize it forming a lupinine citrate salt inside the seeds.

395

In conclusion, the subtle compositional heterogeneity and nutritional quality traits of the

396

examined seed accessions of Lens and Lupinus were explicated through a holistic untargeted



397

approach. The study revealed the most nutritionally valuable seed accessions as follows: the

398

two “Old World lupin Species” Lupinus hispanicus Boiss. & Reutt. (due to its rich

399

compositional profile with free amino, organic acids, and nitrogenous compounds viz. indole-

400

3-acetamide with the latter reported anti-inflammatory activity), and L. angustifolius L.

401

‘Boltensia’ with trace levels of the anti-nutrient quinolizidine alkaloids concurrent with a

402

rather moderate level of amino acids and peptides, and highest sucrose levels. With regards to

403

Lens seeds, the two dark colored Lens culinaris cultivars ‘verte du Puy’ and ‘Black Beluga’

404

were found to possess the most abundant peptide levels and “α-amyrin” triterpene, posing

405

them to likely exhibit antioxidant and anti-inflammatory effects and lacking any alkaloids.

406

It should be noted that the only one examined “New World lupin Species” Lupinus

407

polyphyllus Lindl. ‘Russell’ was revealed to possess the highest levels of toxic alkaloids and

408

least nutritive and deprioritizing it for further breeding programs. The presence of such anti-

409

nutrients in large quantities might have a negative impact on seeds palatability and moreover

410

decrease their human consumption, aside from their reported neurotoxicity.

411

Chemometric tools employed herein were found efficient in pointing out to the species-specific

412

metabolite markers, for example triterpene was regarded as being differentiating metabolites for

413

Lens culinaris specimens versus lupinine alkaloid abundance in Lupinus.

414

Differentiation of the “New World lupine Species” L. polyphyllus Lindl. ‘Russell’ from the

415

other three closely related Old World members of the genus Lupinus, was not achieved

416

suggesting that the metabolic differences arising from the geographical distribution might be

417

obscured due to the recent domestication and/or cultivation traits. We do admit that our

418

selection of Lupinus and Lens resources only covers a small subset of their worldwide

419

diversities, but our approach is certainly feasible for analyzing samples from such further

420

sources. Our approach can also be further applied for exploring other factors on legume


Page 18 of 42

Page 19 of 42


421

metabolites composition, as for instance, seasonal variation, processing method and or

422

storage conditions.

423

ACKNOWLEDGMENTS

424

Prof. Mohamed A. Farag wishes to thank the American University of Cairo Research Support

425

Grant (RSG1-18) and Alexander von Humboldt foundation, Germany for the financial

426

support.

427

CONFLICT OF INTEREST

428

Authors declare no actual or potential conflict of interest including any financial, personal or

429

other relationships with other people or organizations.

430

REFERENCES

431

1.

432

(Lupinus albus L. and Lupinus luteus L.) after extraction of α-galactosides. Food Chemistry

433

2006, 98 (2), 291-299.

434

2.

435

Escudero, N. L., Evaluation of the nutritional quality of the grain protein of new amaranths

436

varieties. Plant Foods for Human Nutrition 2015, 70 (1), 21-26.

437

3.

438

Industrial Research 2014, 3 (6), 285-290.

439

4.

440

(2), 67-82.

441

5.

442

(Leguminosae) inferred from nucleotide sequences of the rbcL gene and ITS 1+ 2 regions of

443

rDNA. Plant Systematics and Evolution 1997, 208 (3-4), 139-167.

444

6.

445

crop plants—biology, production and utilization’.(Eds JS Gladstones, C Atkins, J Hamblin)

446

pp. 1–40. Cambridge University Press: Cambridge: 1998.

Martínez-Villaluenga, C.; Frías, J.; Vidal-Valverde, C., Functional lupin seeds

Aguilar, E. G.; de Jesús Albarracín, G.; Uñates, M. A.; Piola, H. D.; Camiña, J. M.;

Bora, P., Anti-nutritional factors in foods and their effects. Journal of Academia and

Duranti, M., Grain legume proteins and nutraceutical properties. Fitoterapia 2006, 77

Käss, E.; Wink, M., Molecular phylogeny and phylogeography of Lupinus

Gladstones, J., Distribution, origin, taxonomy, history and importance. In ‘Lupins as



447

7.

Khan, M. K.; Karnpanit, W.; Nasar‐Abbas, S. M.; Huma, Z. e.; Jayasena, V.,

448

Phytochemical composition and bioactivities of lupin: a review. International Journal of

449

Food Science and Technology 2015, 50 (9), 2004-2012.

450

8.

451

Lupinus angustifolius L. alkaloid extract. Phytochemistry Reviews 2007, 6 (1), 197-201.

452

9.

453

and their free radical scavenging activity. Natural Product Research 2011, 25 (17), 1641-

454

1649.

455

10.

456

Kachlicki, P., LC-MSMS profiling of flavonoid conjugates in wild mexican lupine, Lupinus

457

reflexus. Journal of Natural Products 2010, 73 (7), 1254-1260.

458

11.

459

P.; Marczak, Ł.; Kachlicki, P.; Stobiecki, M., Structural analysis and profiling of phenolic

460

secondary metabolites of Mexican lupine species using LC–MS techniques. Phytochemistry

461

2013, 92, 71-86.

462

12.

463

compounds of lentil (Lens culinaris Medik): A review. International Journal of Food

464

Properties 2017, 1-15.

465

13.

466

Development of a fast extraction method and optimization of liquid chromatography–mass

467

spectrometry for the analysis of phenolic compounds in lentil seed coats. Journal of

468

Chromatography B 2014, 969, 149-161.

469

14.

470

A.; Pegg, R. B., Free radical-scavenging capacity, antioxidant activity, and phenolic

471

composition of green lentil (Lens culinaris). Food Chemistry 2010, 121 (3), 705-711.

472

15.

473

T.; Forster, J. W.; Kaur, S., Assessment of genetic variation within a global collection of

474

lentil (Lens culinaris Medik.) cultivars and landraces using SNP markers. BMC Genetics

475

2014, 15 (1), 150.

476

16.

477

Comparative metabolite profiling and fingerprinting of genus Passiflora leaves using a

Erdemoglu, N.; Ozkan, S.; Tosun, F., Alkaloid profile and antimicrobial activity of

Zhang, Z.; Yuan, W.; Wang, P.; Grant, G.; Li, S., Flavonoids from Lupinus texensis

Stobiecki, M.; Staszków, A.; Piasecka, A.; Garcia-Lopez, P. M.; Zamora-Natera, F.;

Wojakowska, A.; Piasecka, A.; García-López, P. M.; Zamora-Natera, F.; Krajewski,

Shahwar, D.; Bhat, T. M.; Ansari, M.; Chaudhary, S.; Aslam, R., Health functional

Mirali, M.; Ambrose, S. J.; Wood, S. A.; Vandenberg, A.; Purves, R. W.,

Amarowicz, R.; Estrella, I.; Hernández, T.; Robredo, S.; Troszyńska, A.; Kosińska,

Lombardi, M.; Materne, M.; Cogan, N. O.; Rodda, M.; Daetwyler, H. D.; Slater, A.

Farag, M. A.; Otify, A.; Porzel, A.; Michel, C. G.; Elsayed, A.; Wessjohann, L. A.,


Page 20 of 42

Page 21 of 42


478

multiplex approach of UPLC-MS and NMR analyzed by chemometric tools. Analytical and

479

bioanalytical chemistry 2016, 408 (12), 3125-3143.

480

17.

481

Phytochemical, phylogenetic, and anti-inflammatory evaluation of 43 Urtica accessions

482

(stinging nettle) based on UPLC–Q-TOF-MS metabolomic profiles. Phytochemistry 2013,

483

96, 170-183.

484

18.

485

analysis of six Nigella species seeds via UPLC-qTOF-MS and GC–MS coupled to

486

chemometrics. Food Chemistry 2014, 151, 333-342.

487

19.

488

Hypericum perforatum (St. John's Wort) preparations via UPLC-qTOF-MS and

489

chemometrics. Planta Medica 2012, 78 (05), 488-496.

490

20.

491

A.; Boschin, G., The artificial intelligence-based chemometrical characterisation of

492

genotype/chemotype of Lupinus albus and Lupinus angustifolius permits their identification

493

and potentially their traceability. Food Chemistry 2011, 129 (4), 1806-1812.

494

21.

495

fingerprinting of medicinal licorice roots using a multiplex approach of GC–MS, LC–MS and

496

1D NMR techniques. Phytochemistry 2012, 76, 60-72.

497

22.

498

Quality and Agronomic Characteristics. Crop Science 2002, 42 (3), 979-981.

499

23.

500

Aritsuka, T.; Kaminogawa, S.; Hachimura, S., Dietary melibiose regulates Th cell response

501

and enhances the induction of oral tolerance. Bioscience, Biotechnology and Biochemistry

502

2007, 71 (11), 2774-2780.

503

24.

504

improvement by modulating gut microbiota: the concept revisited. International Journal of

505

Current Microbiology and Applied Sciences 2014, 3 (3), 410-420.

506

25.

507

saccharides enhance net calcium transport from the epithelium of the small and large intestine

508

of rats in vitro. The Journal of Nutrition 2001, 131 (12), 3243-3246.

Farag, M. A.; Weigend, M.; Luebert, F.; Brokamp, G.; Wessjohann, L. A.,

Farag, M. A.; Gad, H. A.; Heiss, A. G.; Wessjohann, L. A., Metabolomics driven

Farag, M. A.; Wessjohann, L. A., Metabolome classification of commercial

Coïsson, J. D.; Arlorio, M.; Locatelli, M.; Garino, C.; Resta, D.; Sirtori, E.; Arnoldi,

Farag, M. A.; Porzel, A.; Wessjohann, L. A., Comparative metabolite profiling and

Muehlbauer, F. J., Carbohydrates in Grain Legume Seeds: Improving Nutritional

Tomita, K.; Nagura, T.; Okuhara, Y.; Nakajima-Adachi, H.; Shigematsu, N.;

Bandyopadhyay, B.; Mandal, N. C., Probiotics, Prebiotics and Synbiotics-in health

Mineo, H.; Hara, H.; Kikuchi, H.; Sakurai, H.; Tomita, F., Various indigestible



509

26.

Panda, H., The Complete book on fruits, vegetables and food processing. NIIR Project

510

Consultancy Services: 2013.

511

27.

512

in beta cells mediates fructose-induced potentiation of glucose-stimulated insulin secretion.

513

Proceedings of the National Academy of Sciences 2012, 109 (8), E524-E532.

514

28.

515

cold hardiness in lentil. Pakistan Journal of Biological Sciences 2000, 3, 2026-2029.

516

29.

517

compounds in lupin flour. Journal of the Science of Food and Agriculture 2009, 89 (14),

518

2421-2427.

519

30.

520

in desiccation tolerance of yellow lupin seeds during maturation and germination. Seed

521

Science Research 1997, 7 (2), 107-116.

522

31.

523

and phenolic compounds of Ziziphus mauritiana fruit. European Food Research and

524

Technology 2005, 221 (3-4), 570-574.

525

32.

526

Chemistry 1996, 57 (1), 51-55.

527

33.

528

Pacific Journal of Clinical Nutrition 1999, 8 (1).

529

34.

530

a bulk oil model system. Journal of Agricultural and Food Chemistry 1995, 43 (6), 1450-

531

1454.

532

35.

533

A.; Abdel-Hamid, A., Rats’ urinary metabolomes reveal the potential roles of functional

534

foods and exercise in obesity management. Food and Function 2017, 8 (3), 985-996.

535

36.

536

Academic Press: 2016.

537

37.

538

Lens. Phytochemistry 2001, 58 (2), 281-289.

Kyriazis, G. A.; Soundarapandian, M. M.; Tyrberg, B., Sweet taste receptor signaling

Ali, A.; Stushnoff, C.; Johnson, D., Negative association of endogenous sorbitol with

Bader, S.; Czerny, M.; Eisner, P.; Buettner, A., Characterisation of odour‐active

Górecki, R.; Piotrowicz-Cieślak, A.; Lahuta, L.; Obendorf, R., Soluble carbohydrates

Muchuweti, M.; Zenda, G.; Ndhlala, A. R.; Kasiyamhuru, A., Sugars, organic acid

Frankel, E. N., Antioxidants in lipid foods and their impact on food quality. Food

Noonan, S.; Savage, G., Oxalate content of foods and its effect on humans. Asia

Koga, T.; Terao, J., Phospholipids increase radical-scavenging activity of vitamin E in

Farag, M. A.; Ammar, N.; Kholeif, T.; Metwally, N.; El-Sheikh, N.; Wessjohann, L.

Nadathur, S.; Wanasundara, J. P. D.; Scanlin, L., Sustainable Protein Sources.

Rozan, P.; Kuo, Y.-H.; Lambein, F., Amino acids in seeds and seedlings of the genus


Page 22 of 42

Page 23 of 42


539

38.

Torino, M. I.; Limón, R. I.; Martínez-Villaluenga, C.; Mäkinen, S.; Pihlanto, A.;

540

Vidal-Valverde, C.; Frias, J., Antioxidant and antihypertensive properties of liquid and solid

541

state fermented lentils. Food Chemistry 2013, 136 (2), 1030-1037.

542

39.

543

peptides. Critical Reviews in Food Science and Nutrition 2008, 48 (5), 430-441.

544

40.

545

F. B. C., beta-Amyrin and alpha-amyrin acetate isolated from the stem bark of Alstonia

546

boonei display profound anti-inflammatory activity. Pharmaceutical Biology 2014, 52 (11),

547

1478-1486.

548

41.

549

tocopherol content and fatty acid profile of selected seeds, grains, and legumes. Plant Foods

550

for Human Nutrition 2007, 62 (3), 85-91.

551

42.

552

contents of phytic acid and divalent cations in low phytic acid (lpa) mutants of rice and

553

soybean. Journal of Food Composition and Analysis 2009, 22 (4), 278-284.

554

43.

555

based foods? Journal für Ernährungsmedizin 2006, 8 (3), 18-28.

556

44.

557

the effects of sparteine, lupanine and lupin extract on the central nervous system of the

558

mouse. Journal of Pharmacy and Pharmacology 1998, 50 (8), 949-954.

559

45.

560

Characteization of Bitter and Sweet Forms of Lupinus angustifolius, an Experimental Model

561

for Study of Molecular Regulation of Quinolizidine Alkaloid Biosynthesis. Chemical and

562

Pharmaceutical Bulletin 2000, 48 (10), 1458-1461.

563

46.

564

nutrition: a review. Mediterranean Journal of Nutrition and Metabolism 2013, 6 (1), 3-16.

565

47.

566

N. Y.; Clawson, D. K.; Hartley, L. W.; Johnson, L. M.; Jones, N. D., Indole inhibitors of

567

human nonpancreatic secretory phospholipase A2. 1. Indole-3-acetamides. Journal of

568

Medicinal Chemistry 1996, 39 (26), 5119-5136.

Elias, R. J.; Kellerby, S. S.; Decker, E. A., Antioxidant activity of proteins and

Okoye, N. N.; Ajaghaku, D. L.; Okeke, H. N.; Ilodigwe, E. E.; Nworu, C. S.; Okoye,

Ryan, E.; Galvin, K.; O’connor, T.; Maguire, A.; O’brien, N., Phytosterol, squalene,

Frank, T.; Habernegg, R.; Yuan, F.-J.; Shu, Q.-Y.; Engel, K.-H., Assessment of the

Greiner, R.; Konietzny, U.; Jany, K., Phytate-an undesirable constituent of plant-

Pothier, J.; CHEAV, S. L.; Galand, N.; Dormeau, C.; Viel, C., A comparative study of

Hirai, M. Y.; Suzuki, H.; Yamazaki, M.; Saito, K., Biochemical and Partial Molecular

Takruri, H. R.; Issa, A. Y., Role of lentils (Lens culinaris L.) in human health and

Dillard, R. D.; Bach, N. J.; Draheim, S. E.; Berry, D. R.; Carlson, D. G.; Chirgadze,

569



Page 24 of 42

570

571

Tables & Figures

572

Table 1: Origin of Lens and Lupinus seed accessions used for metabolites analyses. All accessions

573

are permanently stored at the Department of Bioarchaeology at the Austrian Archaeological

574

Institute (ÖAI), and are accessible on request. Seed providers: Botanischer Garten der Universität

575

Basel, Switzerland (BAS), Jardim Botânico da Universidade de Coimbra, Portugal (COI),

576

Hohenheimer Gärten, Germany (HOH), Alpengarten Patscherkofel und Botanischer Garten

577

Innsbruck, Austria (IB), MARAP HandelsgesmbH, Vienna, Austria (MRP), and Vollkraft

Country of

ÖAI

origin

accession

Sample Species and cultivar

Provider

code number LCB

Lens culinaris Medik. subsp. culinaris ‘verte du Puy’ (brown

Austria VKR

2827

colored seed)

LCO

Lens culinaris Medik. subsp. culinaris ‘Petite Rouge d’Egypte’

MRP

Austria

2376

LCY

Lens culinaris Medik. subsp. culinaris (yellow colored seed)

BAS

Switzerland

555

Lens culinaris Medik. subsp. culinaris ‘Black Beluga’

MRP

Austria

2375

LU_PO

Lupinus polyphyllus Lindl. ‘Russell’

HOH

Germany

510

LU_A

Lupinus angustifolius L. ‘Boltensia’

IB

Austria

649

LU_L

Lupinus luteus L.

IB

Austria

1325

LU_H

Lupinus hispanicus Boiss. & Reutt.

COI

Portugal

2872

LCBK

578

Naturnahrung Handels und Produktions GmbH, Grimmenstein, Austria (VKR).


Page 25 of 42


579 580



Page 26 of 42

Table 2: Relative percentile of silylated primary and secondary metabolites in Lens and Lupinus seed accessions as analyzed via GC/MS n= 3. Different letters indicate significant differences between seed specimens according to least significant difference analysis (LSD)

Peak

RT

#

(min)

L.culinaris, KI

Metabolites

brown colored (a)

L. culinaris, Petite Rouge d’Egypte (b)

L.culinaris,

L.culinaris

yellow

Black

colored (c)

Beluga (d)

0.6

0.1

L.polyphyllus (e)

L.angustifolius (f)

L.luteus (g)

L.hispanicus (h)

0.1

0.4

0.1

Acids

G1

5.65

1019

Carbamic acid (TMS) Lactic

G2

6.39

1055

acid(2TMS)oxy -, ester Methyl 2-ethyl

G3

6.66

1067

malonate (TMS)

G4

6.84

1076

G5

9.87

1203

G6

10.77

1238

G7

12.73

1315

Acetic acid (2TMS) Malonic acid

0.3

0.1 (0.01)

b

(0.04)a,c,d,e,f,g, h

(0.6)

b

(0.01)

0.1 b

(0.02)

b

(0.03)

b

b

(0.2)

(0.01)b

0.2

0.3

0.2

0.2

0.2

0.2

0.3

0.2

(0.1)g

(0.02)

(0.1)g

(0.02)g

(0.04)g

(0.0)

(0.2)a,c,d,e,h

(0.01)g

tr.

tr.

tr.

tr.

0.2

tr.

tr.

tr.

e

e

e

e

e

-e

-

-

1.2

0.1 (0.01)

-

b

(1.01)a,c,d,e,f,g, h

b,c,d.f.g.h

-

0.1

(0.1)

0.1

(0.03)

b

(0.01)

0.1 b

-

-

0.1

(0.01)

b

0.2

b

0.1 b

(0)

(0.1)

(0.01)b

0.4

0.2

0.14

0.3

1.8

0.2

0.2

0.6

(0.1)e

(0)e

(0.1)e

(0.1)e

(0.8)a,b,c,d,f,g,h

(0.03)e

(0.04)e

(0.3)e

0.2

2.1

0.3

0.4

0.4

0.3

0.2

0.2

oxy (3TMS)

(0.1)

(0.3)

(0.1)

(0.03)

(0.1)

(0.1)

(0.04)

(0.01)

Succinic acid

0.1

1.0

0.3

0.1

0.2

0.3

0.6

0.5

(2TMS) Carbamic acid,

(2TMS)

(0.04)

b

(0.4)

a,c,d,e,f,g,h

(0.2)

b

(0.01)

b


b

(0.2)

(0.01)

b

b

(0.3)

(0.04)b

Page 27 of 42


G8

12.99

1324

G9

13.64

1350

G10

14.92

1399

Methyl succinic

0.1

0.8

0.1

0.1

0.4

0.1

0.2

0.1

acid (2TMS)

(0.01)b

(0.1)a,c,d,f,h

(0.04)b

(0.01)b

(0.6)

(0)b

(0.2)

(0.02)b

Fumaric acid

tr.

tr.

tr.

tr.

tr.

tr.

tr.

-

-

-

-

-

-

-

-

tr.

tr.

-

-

(2TMS)

-

-

Methylmaleic

tr.

acid (2TMS)

-

tr.

-

-

0.2

0.51

tr.

0.2

tr.

tr.

0.2

0.3

(0.04)

(0.4)e,c,f

-b

(0.01)

-b

-b

(0.1)

(0.1)

tr.

0.1

tr.

0.1

tr.

tr.

0.1

0.1

-

g

-

3.3

1.8

-

3,4G11

15.72

1431

Dihydroxybuta noic acid (3TMS)

G12

16.60

1467

G13

17.08

1486

G14

18.40

1541

G15

18.85

1560

Hydroxymaloni c acid (3TMS) Malic acid (3TMS) L-Threonic acid (3TMS) L-Threonic acid (3TMS) α-

G16

19.19

1574

Hydroxyglutari c acid (3TMS)

G17

19.78

1598

-

(0.01)

1.4 (0.1)

c,f

0.9 e

tr. 0.2 (0.01) tr. -

(0.1)

-

b,g

(0)

1.1 e

-

-

-

(0.3)

1.4 e

(0.2)

e

(1.1)

a,b,c,d,f,g

(0.3)

(0.04)

c,f

0.8 e

(0.5)

(0.01) 2.3

e

(0.3)

tr.

tr.

tr.

tr.

tr.

tr.

-

-

-

-

-

-

0.2

0.3

0.1

0.1

0.1

(0.03)

(0.14)

(0)

0.1 (0.1)

e

c

(0.01)

(0.13)

tr.

tr.

0.1

tr.

0.1

0.1

-

-

(0.02)

-

(0.2)

(0.01)

3-Hydroxy-3-

0.1

0.3

0.1

0.1

0.1

0.1

0.5

0.12

methylglutaric

(0.04)

(0.2)

(0.02)

(0.01)

(0.1)

(0.02)

(0.8)

(0.1)



Page 28 of 42

acid (3TMS) G18

23.11

1748

G19

24.56

1815

Trans-Aconitic acid (3TMS) Citric acid (4TMS)

Total acids

0.1 (0.03)

h

3.8

0.1

-

(0.1)

0.01

(0.5)

(0)

0.1 h

1.6

e,h

(1.4)

e,h

0.1

0.1

(0.02)

(0.1)

(0.03)

3.0

6.7

5.1

(0.1)

b,c,g

(1.4)

h

tr.

0.2

-

(0.03)a,c,f

1.18

(0.01)

(2.03)

e,h

6.9 (0.68)b,c,g

6.9

7.6

4.6

6.3

14

8.5

4.9

11.7

tr.

tr.

1.4

tr.

tr.

tr.

tr.

tr.

-

-

(0.3)f

-

-

-c

-

-

0.5

1.3

0.1

0.1

0.3

0.6

1.2

0.3

(0.4)g

(0.1)c

(0.1)g,b

(0.1)

(0.04)g

(0.5)g

(1.4)a,c,e,f,h

(0.1)g

tr.

tr.

tr.

tr.

tr.

tr.

c

b

Alcohols G20

5.28

1001

G21

7.88

1122

G22

G23

10.88

11.66

1242

1273

1,2-Propanediol (2TMS) Ethylene glycol (2TMS) Diethylene

0.1

tr.

(0.01)a,c,d,e,f,g,

b,c

glycol (2-TMS)

-

Glycerol

0.2

0.1

(3TMS)

(0.1)

(0)

h

a,b,d,f,h

-

b,c

-

b

-

-b,c

-

-

1.1

0.3

0.2

0.3

0.6

1.6

(0.4)

(0.01)

(0.04)

(0.1)

(0.5)

(0.3)

0.4

0.03

0.1

0.1

0.1

0.2

1,2G24

22.02

1698

Propanediol-1phosphate

0.1 (0.03)

c

-

(0.4)

a,e,f,g,h

(0)

c

(0.1)

c

(0.01)

c

(0.2)

c

(0.04)c

(3TMS) GlycerylG25

33.85

2284

glycoside (TMS)

1.4 b,c,f,g,h

(0.2)

0.04 (0)

a,d

0.5 (0.4)

a,d

1.1 (0.01)

b,e,f,g,h


0.1 (0.03)

0.1 a,d

(0)

a,d

0.1 (0.1)

a,d

0.1 (0.01)a,d

Page 29 of 42


Total alcohols

2.2

1.5

3.5

1.9

0.7

1.2

2.0

2.2

Lupinine

-

-

-

-

tr.

tr.

0.2

6.2

-

-

(0.2)

(0.7)

Lupinine

tr.

tr.

h

-

-

h

tr.

tr.

tr.

4.3

7.3

h

-

h

-

h

-

(4.3)

tr.

tr.

tr.

tr.

7.9

tr.

0.1

tr.

-e

-e

-e

-e

(2.2)a,b,c,d,f,g,h

-e

(0.2)e

-e

tr.

tr.

0.3

-

-e

-e

(0.2)a,c,d

-

-

Alkaloids G26

17.08

1476

G27

17.47

1502

G28

32.63

2222

G29

34.99

2342

G30

35.76

2381

isomer α-Isolupanine

-

37.80

2485

(0.8)a,b,d,e,f,g

α-Isolupanine

tr.

isomer

-e

13-Hydroxy-

tr.

tr.

tr.

tr.

10.3

0.1

tr.

tr.

lupanine (TMS)

-e

-e

-e

-e

(3.2)a,b,c,d,f,g,h

(0.1)e

-e

-e

-

-

-

-

0.4

tr.

(0.2)

-

-

-

tr.

tr.

tr.

tr.

18.9

0.2

4.6

13.5

0.2

0.5

-

13-HydroxyG31

h

lupanine (TMS) isomer

Total alkaloids

Amino acids G32

7.42

1103

G33

10.13

1213

L-Alanine, N(2TMS) L-Valine, N(2TMS)

0.3 (0.1)

0.2 h

(0.04)

0.1

tr.

(0.04)

h,d

-

0.3 h

(0.2)

0.3 h

0.1 (0.1)

(0.01)

h

0.1 (0)

b


(0.1)

h

(0.1)

0.2 h

(0.2)

1.2 h

(0.2)a,b,c,d,e,f,g

0.1

0.1

0.1

0.1

(0.03)

(0)

(0.1)

(0.03)b


G34

11.21

1255

G35

11.46

1265

G36

12.14

1291

G37

12.42

1302

G38

14.49

1383

G39

15.48

1422

G40

15.53

1424

G41

17.84

1517

Serine (2TMS) Pipecolic acid

18.44

1543

19.40

1583

G44

19.66

1594

tr.

tr.

tr.

tr.

0.1

0.1

-

(0.2)

-

-

-

-

(0.1)

(0.01)

tr.

tr.

tr.

tr.

0.8

0.1

0.1

0.8

e,h

e,h

e,h

e,h

L-Threonine

Glycine (3TMS)

tr.

tr.

tr.

tr.

0.1

tr.

0.1

g,h

h

-

h

-

h

-

h

b

-

(0.02)a,b,c,d,e,f

0.2

0.2

0.1

0.3

0.2

0.5

(0.01)

(0.2)

(0.2)b,e

0.1 (0.03)

L-Threonine

tr.

(3TMS)

-

l-Aspartic acid

0.4

(2TMS)

(0.1)

L-lysine

tr.

(3TMS)

-

L-Proline, 5oxo-1-(2TMS)

Phenylalanine

L-Asparagine (2TMS) L-Asparagine (2TMS)

f

h

(0) h

(0.1)

(0.1)a,b,c,d,f,g

h

0.2

(0.02)

h

tr.

-

(0.1)

e,h

-

-

-

a,b,c,d,f

-

(0.1)

(TMS) G43

0.3

-

LG42

tr.

(TMS)

(TMS)

Page 30 of 42

(0.1)

(0.02)

(0.04)

tr.

tr.

tr.

tr.

tr.

tr.

tr.

-

-

-

-

-

-

-

tr.

0.7

h,d

(0.3)c,e,f,g

0.2

-

(0.3)

-

0.5 h

(0.02)

0.2 g

(0.2)

0.2 h

(0.02)

h

-

tr.

tr.

tr.

tr.

tr.

0.1

-

-

-

-

-

(0.02)

1.4

0.01

0.3

0.7

0.4

1.7

0.2

0.4

(1.2)

(0)

(0.3)

(0.1)

(0.2)

(0.4)

(0.2)

(0.1)

0.1

tr.

tr.

tr.

tr.

0.5

h

h

h

h

-

(0.4)a,c,d,e,f,g

0.1

0.1

(0.1)

(0.1)b

tr.

0.2

a

(0.3)

0.1 (0.01)

h

0.1 (0.01) 0.2 (0.13)

b,e,g

-

(0.1)

0.6 (0.2)

c,d,e,f,h

0.01 (0)

a

h

-

-

0.1

0.1

b

b

(0)

0.1 (0.1)

(0)

0.1 (0.02)

0.2

tr.

(0.01)

a


-

-

0.1 b

(0.01) -

b

-

Page 31 of 42


G45

20.21

1618

G46

21.30

1666

Glutamic acid (3TMS)-I L-Asparagine (2TMS)

Total amino acids

0.6

-

(0.6) 0.2

-

(0.2)

0.2

1.04

0.2

0.2

(0.2)d

(0.2)c,e,f,h

(0.2)d

(0.2)d

tr.

0.2

-

(0.01)

-

-

tr.

-

-d

tr.

tr.

-

-

3.4

1.2

1.6

3.3

2.2

3.3

0.9

4.8

0.5

0.8

0.1

0.2

0.1

0.2

1

0.2

(0.6)

(0.1)

(0.1)

(0.01)

(0.01)

(0.03)

(0.9)

(0.04)

tr.

tr.

tr.

tr.

tr.

tr.

a

-

a

-

a

-

-

a

a

-

-

0.1

0.2

0.2

0.2

1

0.2

0.5

0.3

0.3

0.4

0.4

Aromatic Benzaldehyde, G47

13.81

1357

4-hydroxy, oxime (2TMS) p-

G48

20.34

1623

Hydroxybenzoi c acid (TMS)

Total aromatics

0.1 (0.04)

-

c,d,e,f,g

0.6

0.8

0.2

0.1

Fatty acids Hexadecanoic G49

29.12

2043

acid (TMS) Linoleic acid

G50

32.11

2199

(TMS) Oleic acid

G51

32.22

2201

(TMS) Stearic acid

G52

32.69

2224

(TMS)

G53

34.36

2310

Oxylipin

(0.04)

(0.01)

0.1 (0.02)

tr. g,h

c,g,h

-

0.2 (0.02)

tr. g,h

0.1 (0.01) -

g,h

g,h

(0.4)

(0.03)

(0.1)

(0)

(0.4)

(0.04)b

0.3

0.2

0.2

0.2

0.3

0.7

b,h

(0.2)

0.5

c,g,h

(0.3)

0.04

0.1

-

(0) -

g,h

0.7

0.2 b

(0.03) -

(0.01)

h

0.3

(0.02)

h

0.1 g,h

(0.01)

(0.03)

g,h

(0.1)

0.3

g,h

0.1 g,h

-


(0.04) 0.4

(0.1)

g,h

(0.1)

g,h

0.1 g,h

(0) -

g,h

b

(0.2)

a,b,e,f

0.4 (0.3)

a,b,e,f

0.2 (0.2)

a,b,c,d,e,f

-

(0.04)a,b,c,d,e,f 0.6 (0.04)a,b,d,e,f 0.3 (0.02)a,b,c,d,e,f -


Page 32 of 42

(0.2) 1Monopalmitin G54

38.27

2508

(TMS)

Total fatty acids

0.1 (0.01)

b

0.6

tr.

tr.

0.1

a,c,d,e

b,f

-

(0)

b

0.2

1.4

0.9

1.3

tr.

0.5

2.2

0.5

-

0.1 (0.02)

0.1 b

tr.

tr.

-

-

1.04

1.4

2.3

1.1

0.4

(0.02)

c

Inorganics Phosphoric acid G55

9.18

1175

(2TMS)

d

(0.2)

(0.6)

(0.3)b

5.8

1.5

2.4

5.7

9.7

(3.4)a,b,d,e,f

(0.2)c,g

(0.8)b,c,g

(0.03)c,h

(7.2)b,d,h

(2)a,b,e,f,g

0.1

4.1

8.01

2

3.5

6.03

11.1

0.3

2.3

0.3

0.7

0.3

1.1

0.9

0.6

(2TMS)

(0.3)b

(0.7)a,c,d,e,f,h

(0.3)b

(0.1)b

(0.1)b

(0.3)b

(1.01)

(0.1)b

(3TMS)

0.1

2.2

0.1

0.2

0.2

0.42

0.3

0.3

(0.1)b

(0.4)a,c,d,e,f,g,h

(0.1)b

(0.02)b

(0.12)b

(0.2)b

(0.5)b

(0.02)b

tr.

tr.

tr.

0.1

tr.

tr.

-

d

d

-

d

-

(3TMS) 11.57

1269

phosphate

Total inorganics

d,h

(0.4)

3.7

0.1

3.6

(0.6)c,h

(0)c,e,g,h

4.8

(0.5)

-

d

(0.1)

a,b,c,e,f,g

d,b

d

1.4

(0.3)

monomethyl

G56

1.1

d

Nitrogenous compounds G57

7.17

1091

G58

7.65

1112

G59

10.99

1247

Ethanolamine

hydroxylamine Urea (2TMS) Silanamine

G60

11.40

1262

trimethyl-N(TMS)-N-[2-

d

-

0.1 (0.01)h

-

-

(0.01)

a,c,e,f,h

-

tr. -d

tr.

0.1

0.1

0.2

0.2

0.6

-h

(0)h

(0.04)h

(0.01)h

(0.3)h

(0.2)a,c,d,e,f,g


Page 33 of 42


[(TMS)oxy]eth yl]G61

13.39

1340

G62

13.90

1359

Phenylethanola mine (3TMS) Pipecolinic acid (2TMS) α-

G63

14.56

1385

Aminoisobutyri c acid (3TMS)

G64

19.52

1588

0.1

0.1

0.4 b

(0.1)

(0.1)

a,c,d,e,f,g

(0.1)

0.01

-

(0)

0.1 b,g

(0.1)

-

h

a,c,d,e,f,g,h

(0.02)

0.1 b

b,g

(0.1)

-

0.1 b

22.91

1739

(0.1)

(0.1)

(0.01)

0.01

0.1

(0)

0.2 b

0.1 b

h

(0.01)b,e,g

0.2

(0.02)

b

(0.2)

0.1

a,b,c

(0.01)b

Cadaverine

tr.

0.3

tr.

0.1

tr.

0.1

0.1

0.1

(4TMS)

-b

(0.1)a,c,d,e,f,h

-b

(0.01)b

-b

(0.02)b

(0.1)

(0.01)b

-

-

-

-

-

-

0.2

0.2

1H-Indole-3G65

(0.02)

0.1 b

-

h

0.1 (0.01)

(0.03)

0.2 b

tr.

-

0.1

0.4 (0.1)

0.2 b

acetamide

0.1 (0.02)

(TMS) G66

38.18

2504

Uridine (TMS)

G67

18.03

1525

GABA (3TMS)

Total nitrogenous compounds

(0) 0.6 (0.4) 1.5

(0.1)

0.3 h

5.9

(0.04)

d,f,g,h

0.1 (0.01)

h

0.2 c

(0.02)

0.2 h

(0.1)

9.3 (1.3)e

0.1 c

(0.03)

0.1 c

(0.01)b,c,e

0.3

0.7

0.4

0.3

0.5

1.1

(0.3)

(0.02)

(0.32)

(0.01)

(0.8)

(0.2)

1.2

2.2

1.4

2.6

2.4

12.4

tr.

tr.

tr.

-a,d

-c,e

-a,d

-

-

-

0.5

1.3

0.7

0.5

0.3

0.2

Peptides G68

17.73

1513

G69

18.15

1531

Cys-Gly

tr.

(3TMS)

-c,e

Glycyl-l-

1.1

0.01



glutamic acid

Page 34 of 42

(0.8)

(0)d

(0.5)

(0.1)b

(0.6)

(0)

(0.42)

(0.04)

0.6

tr.

0.4

0.5

tr.

tr.

0.1

0.2

(2TMS) G70

21.82

1689

G71

25.69

1870

Ser-Leu (2TMS)

(0.1)

c,d,g,h

-

(0.3)

a

(0.02)

a,e,g

-

-

(0.04)

a,d

(0.1)a

Ala-Thr

0.1

tr.

0.1

0.2

tr.

tr.

tr.

tr.

(2TMS)

(0.03)

-

(0.1)

(0)

-

-

-

-

1.7

0.1

1.02

1.9

0.7

0.6

0.4

0.4

0.1

tr.

0.1

0.1

tr.

0.1

0.1

0.1

(0.01)b

-a,c,d,g

(0)b

(0.02)b,e,f,g

-d,g

(0.03)d

(0.03)b,d,e

(0.01)

5.8

2.2

3.2

4.1

2.03

0.9

3.3

1.3

(0.3)e,f,g,h

(0.2)

(2.9)

(0.4)

(1.6)a

(0.2)a

(2.9)a

(0.01)a

5.9

2.2

3.3

4.2

2.1

1

3.4

1.3

tr.

0.06

tr.

tr.

tr.

tr.

tr.

0.1

b,h

b,h

-

h

-

h

-

(0.01)a,c,d,f,g

Total peptides Sterols/triterp enes G72

48.63

3036

G73

49.82

3096

β-Sitosterol (TMS) α-Amyrin (TMS)

Total sterols/triterpenes Sugars G74

17.38

1498

G75

17.56

1506

G76

19.29

1579

G77

21.91

1693

L-Threitol

b,h

(4TMS)

-

Meso-Erythritol

tr.

(4TMS)

-

Xylulose

tr.

(4TMS) Xylitol (5TMS)

e

-

tr. -

(0.03)

a,c,d

-

-

0.06

tr.

tr.

0.1

tr.

tr.

0.1

(0.03)

h

-

-

(0.03)

-

-

(0.01)c

-

-

-

0.1

tr.

tr.

e

-e

tr.

tr.

0.03

tr.

tr.

tr.

-

-

(0.02)

-

-

-

-


(0.01)

a,f,g

-

-

Page 35 of 42

G78


22.42

1717

D-Arabitol

tr.

tr.

tr.

tr.

0.5

tr.

tr.

0.1

(5TMS)

-

-

-

-

(0.8)

-

-

(0.02)

tr.

tr.

tr.

tr.

-

-

-

-

2-Keto-lG79

23.09

1747

gluconic acid (5TMS)

tr.

-

-

tr.

-

-

α-DG80

24.63

1818

Glucopyranosid e, methyl

17.03 (1.2)

tr.

e,f,g,h

-

13.3 (3.2)

e,f,g,h

17.6 (0.4)

e,g,h

1.2 (0.8)

4.6

a,c,d

(0.3)

a,c

1.8 (1.8)

1.2

a,c,d

(0.2)a,c,d

(4TMS) G81

24.76

1825

G82

26.67

1923

G83

26.89

1930

G84

26.97

1933

G85

27.16

1943

G86

27.24

1947

G87

27.89

1980

D-Pinitol

1.7

(5TMS)

(0.03)

D-Sorbitol

tr.

(6TMS)

-

D-Sorbitol (6TMS) Fructose oxime (6TMS) Fructose oxime (6TMS) Myo-Inositol (6TMS) DGlucoseoxime

-

-

1.1

2.5

1.2

0.3

1.8

0.7

(0.2)g

(0.1)f,h

(0.3)

(0.04)d,g

(2)c,f,h

(0.1)d,g

tr.

tr.

0.3

tr.

0.6

tr.

-

-

(0.5)

-

(1)

-

0.1

0.02

0.1

0.1

0.2

0.1

0.3

0.1

(0.01)

(0)

(0.03)

(0)

(0.2)

(0.01)

(0.4)

(0.1)

tr.

0.1

0.2

tr.

0.2

0.1

0.1 (0.02)

-

g

0.1

-

(0.01) 0.1 (0.02)

-

f,g

tr.

0.3 b

(0.1)

a,g

-

-

(0)

g

(0.3)

0.1

0.1

0.2

0.4

0.4

0.1

(0.02)

(0)

(0.3)

(0.01)

(0.2)

(0.01)

0.1

0.2

0.1

0.7

0.7

0.3

(0)

f,g

0.1 (0.03)

(0)

f,g

0.2 (0.02)


g

f,g

(0.1)

0.2 (0.2)

-

(0.04)

a,c,d,e,g,h

0.1 (0)

(0.2)

(0.4)

a,d,e

a,c,e,f,h

0.2

(0.01)

(0.01)f,g tr.

b

(0.2)

-


Page 36 of 42

(6TMS) G88

28.05

1988

G89

28.43

2008

D-Gluconic acid (6TMS) Glucose oxime (6TMS)

0.1

tr. -

b,f

a,d,g

-

-

-

(0.02) 0.1 (0.02)

0.04

h

g,h

(0.03)

tr.

tr.

-

0.1 h

28.99

2040

methyloxime

-

29.81

2079

G92

34.89

2337

Myo-Inositol (6TMS)

35.41

2362

0.01 (0)

2478

39.00

2546

40.66

2630

(0.2)

e

(0.1)

-

a

-

-

0.6

2.1

(0.3)

a,c,d,h

(0.1)

0.1

tr.

b,h

(0.1) tr.

2.2 (1.1)

-g

-

-

c,h

(0.03)a,b,c,e,g

3.1 h

(0.1)e,f,g

0.2

0.1

tr.

0.2

0.1

acid (5TMS)

(0.01)

(0.1)

(0.1)

(0.1)

(0.01)

-

(0.3)

(0.04)

0.2

tr.

tr.

0.1

tr.

0.1

0.1

a,d,e,h

d

a,d

(0.1)

(0)b,d

phosphate

(0.01)

b,c,d,e,f

-

-

0.3 (0.01)a,b,c,e.f, h

(0.02)

a,b,d

-

0.2

0.2

0.2

0.3

0.1

0.1

0.1

0.1

(0.1)e,f,h

(0.1)h

(0.02)

(0)e,f,g,h

(0.02)a,d

(0.03)a,d

(0.03)d

(0.02)a,b,d

Sucrose

20.1

0.1

23.6

17

15.8

40.2

14.5

8.5

(8TMS)

(2.1)

(0.03)f

(4.3)

(0.1)

(5.1)

(0.8)b

(11.5)

(0.5)

6.5

1.1

7.7

8.04

2.5

1.2

1.3

1.8

Mannobiose

DG96

2.7

tr.

(0.2)

0.2

(8TMS) G95

(0.5)

e,f

0.1

0.7

b,h

0.2

3-α37.66

3.01

(0.02)

0.1 h

0.6

(7TMS)

G94

(0.1)

-

e

b

0.1 h

D-Glucuronic

D-Myo-Inositol G93

2.6

(0.01)

(0.01)

(8TMS) G91

(0) 0.1

Maltose G90

0.1

Glucopyranose, 4-O-(4TMS)-β-

b,d,e,f,g,h

(1.1)

(0.4)

a,e,f,g,h

(1)

e,d,f,g,h

(0.1)

a,c,e


(1.1)

a,b,c,d

(0.4)

a,b,c,d

(0.9)

a,b,c,d

(0.4)a,b,c,d

Page 37 of 42


Dgalactopyranos yl] (4TMS) G97

41.81

2689

G98

42.12

2704

G99

42.43

2719

G100

43.11

2755

G101

43.29

2783

G102

43.80

2790

G103

45.76

2889

G104

49.31

3070

G105

49.36

3085

Total sugars

Galactinol (6TMS) D-Turanose (7TMS) Galactinol (9TMS) D-Lactose (8TMS) D-Lactose (8TMS) Galactinol (9TMS) Melibiose (8TMS) Galactinol

1.7

1.3

(0.3)

g

2.1

(0.5)

0.1

0.7

(0.04)

(0.6)

c,e,f

1.4

2.4

f,g,h

(0.1)

(0.03)

tr.

0.1

f,g,h

0.4

(0.6)

(0.2)

0.1

b

-

(0.01)

(0.01)

b

0.5

c,d

(0.2)

0.9

a,c,d

(0.2)c,d

tr.

0.1

0.1

b

-

(0.01)

(0.02)

0.2

1.9

0.3

0.3

0.4

0.3

0.2

0.2

(0.1)b

(0.9)a,c,d,e,f,g,h

(0.02)b

(0.03)b

(0.2)b

(0.1)b

(0.04)b

(0.1)b

0.3

3

0.3

0.3

0.5

4.7

3.1

1.6

(0.1)

(1.8)

(0.01)

(0.02)

(0.3)

(0.6)

(1.5)

(0.2)

0.5

3.2

0.6

0.5

0.5

4.8

3.1

1.6

(0.1)

(2)

(0.04)

(0.03)

(0.3)

(0.5)

(1.5)

(0.2)

0.7

2.5

0.9

0.6

0.3

0.9

2.1

1.3

(0.3)b

(1.4)a,c,d,e,f,h

(0.1)b

(0.02)b

(0.1)b

(0.1)b

(1.8)

(0.2)b

1

45.9

7.7

0.6

6.2

0.5

17.7

b

(0.2)

(3.1)

a,c,d,e,f,g

(7.4)

b

(0.1)

b

(9.03)

b

b

(0.1)

(15.9)

1.3 b

(0.4)

2.4

10.1

2.9

2.8

3.1

2.9

6.04

2.6

(9TMS)

(2.4)

(1.2)

(1.7)

(1.8)

(3.8)

(0.7)

(2.9)

(0.5)

Unknown

15.7

10.1

14.8

13.9

20.6

13.3

15.9

disaccharide

(2.02) 72.4

b

(6)

a,c,d,e,f,g,h

80.3

(1.4)

b

79.1

(0.8)

b

71

Unknowns


b

(5.5)

b

(1.4)

(12.3)

56.6

78.04

73.1

13.5 b

(11.1)b 40.2


G106

13.07

1328

Unknown

G107

22.53

1721

Unknown

0.1

tr.

0.1

tr.

tr.

tr.

tr.

-

(0.03)d

-

(0.01)b,e,f,g,h

-d

-d

-d

-d

tr.

0.03

-

(0)

-

-

-

Total

a

tr.

tr.

Total unknowns

Page 38 of 42

-

tr. -

0.1

0.1

0.1

0.1

tr.

tr.

tr.

0.1

100

100

100

100

100

100

100

100

(LCB) Lens culinaris Medik subsp. culinaris “verte du Puy” brown colored seed; (LCO) L. culinaris Medik subsp. culinaris “Petite Rouge d’Egypte”; (LCY) L.

culinaris Medik subsp. culinaris yellow colored seed; (LCBK) L. culinaris Medik subsp. culinaris “Black Beluga”; (LU_PO) Lupinus polyphyllus Lindl “Russell”; (LU_A) L. angustifolius L. “Boltensia”; (LU_L) L. luteus L.; (LU_H) L. hispanicus Boiss & Reutt. b

-

(-): absent. c Tr.: Present at trace level < 0.05. Bold numbers: indicate the highest percentile levels of the different seed metabolites. Results are average of 3 independent biological replicates ± (std. deviation).

a–h Different letters indicate significant differences between seed specimens according to least significant difference analysis (P < .05; Tukey's test).


Page 39 of 42

581


Figure Captions

582 583

Figure 1: Major metabolite classes’ percentile levels in different Lens and Lupinus seeds as listed in Table 1 analyzed using GC/MS post silylation.

584 585 586 587 588

Figure 2: GC/MS based unsupervised hierarchical clustering and principal component analyses (PCA) of metabolites found in Lens and Lupinus seeds. (A) HCA plot (B) PCA score plot of PC1 vs. PC2 scores. (C) Loading plot for PC1 & PC2 contributing primary metabolites and their assignments. The metabolome clusters are located at the distinct positions in two-dimensional space described by the principal component 1 (PC1) = 36% and PC2 = 17%.

589 590 591 592

(LCB) Lens culinaris Medik subsp. culinaris “verte du Puy” brown colored seed; (LCO) L. culinaris Medik subsp. culinaris “Petite Rouge d’Egypte”; (LCY) L. culinaris Medik subsp. Culinaris yellow colored seed; (LCBK) L. culinaris Medik subsp. culinaris “Black Beluga”; (LU_PO) Lupinus polyphyllus Lindl “Russell”; (LU_A) L. angustifolius L. “Boltensia”; (LU_L) L. luteus L.; (LU_H) L. hispanicus Boiss & Reutt.

593 594 595 596 597 598

Figure 3: GC/MS based OPLS-DA score plot derived from modelling Lupinus seeds against Lens seeds (A). The S-plot (B) shows the covariance p[1] against the correlation p(cor)[1] of the variables of the discriminating component of the OPLS-DA model. Cut-off values of P

Mass Spectrometry-Based ... - ACS Publications

Recommend Documents