Accepted Manuscript

Carotenoid changes of colored-grain wheat flours during bun-making

Luboš Paznocht, Zora Kotíková, Matyá š Orsák, Jaromír Lachman, Petr

Martinek

PII: S0308-8146(18)31946-0
DOI: https://doi.org/10.1016/j.foodchem.2018.11.019
Reference: FOCH 23827

To appear in: Food Chemistry

Received Date: 28 June 2018

Revised Date: 25 October 2018
Accepted Date: 1 November 2018

Please cite this article as: Paznocht, L., Kotíková, Z., Orsák, M., Lachman, J., Martinek, P., Carotenoid changes of
colored-grain wheat flours during bun-making, Food Chemistry (2018), doi: https://doi.org/10.1016/j.foodchem.
2018.11.019

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1 Carotenoid changes of colored-grain wheat flours during bun-making

3 Luboš Paznochta, Zora Kotíkováa, Matyáš Orsáka, Jaromír Lachmana,*, Petr Martinekb

4 a Department of Chemistry, Faculty of Agrobiology, Food and Natural Resources, Czech

5 University of Life Sciences Prague, Kamýcká 129, 165 00 Prague – Suchdol, Czech Republic

6 b Agrotest Fyto, Ltd., Havlíčkova 2787, 767 01 Kroměříž, Czech Republic

8 *Corresponding author. Tel.: + 420-224382717.

9 E-mail address: [email protected] (J. Lachman).

10 Abstract

11 Colored-grain wheat genotypes were used in the preparation of flour, dough, buns, and buns

12 stored for a short period of time. The main carotenoid in all genotypes was lutein, followed by

13 its esters, zeaxanthin, and -carotene, while antheraxanthin and -carotene occurred only at

14 negligible levels. The highest carotenoid contents were observed in yellow- and purple-

15 grained genotypes. After the preparation of dough, total carotenoid content (TCC) decreased

16 significantly by an average of 61.5%. Zeaxanthin was shown to be stable, whereas -carotene

17 was destroyed. In baked buns, the average decrease of TCC and all-E-lutein was lower than in

18 unbaked dough. Greater decreases were recorded for esters, antheraxanthin, and -carotene.

19 After storing buns for 24 hours at room temperature, approximately one-quarter of TCC

20 observed in the original flour was preserved. Z-Isomers of lutein occurred in minor

21 concentrations, but the degradation of this component, and that of zeaxanthin, was low,

22 suggesting E- to Z-isomerization.

23 Keywords: esterified carotenoids; non-esterified carotenoids; colored-grain wheat genotypes;

24 flour; dough; baked buns; short-term stored buns

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25 Abbreviations: Ba  blue aleurone; BHT  butyl hydroxytoluene; DW  dry weight; Pp 

26 purple pericarp; Red  red grain; TCC  total carotenoid content; TLC  total lutein content;

27 Ye  yellow endosperm

28

29 Chemical compounds

30 -carotene (PubChem CID: 4369188); antheraxanthin (PubChem CID: 5281223);

31 -carotene (PubChem CID: 5280489; lutein (PubChem CID: 5281243); zeaxanthin

32 (PubChem CID: 5280899).

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33 1. Introduction

34 Cereals play an essential role in the human diet, especially wheat, which is the second

35 most produced cereal on earth. Around 750 million metric tons are produced annually,

36 corresponding to a yearly consumption of 67 kg per capita (FAOSTAT, 2018). In Europe,

37 wheat is the most commonly used cereal for the production of flour and its resulting food

38 products  breads, pastries, and pastas. Due to the increasing worldwide prevalence of

39 lifestyle diseases, the overall demand for nutritionally valuable foodstuffs is increasing. In

40 addition to traditional cereal crops, there is also an opportunity for the cultivation and

41 processing of non-traditional modern and ancient cereals such as einkorn, emmer, spelt, and

42 colored-grain wheat. Such crops are valuable not only as a source of essential nutrients, but

43 also as a source of a wide range of biologically active compounds, including anthocyanins,

44 carotenoids, and phenolic acids. These compounds are notable for their biological functions,

45 particularly their high antioxidant activity, thanks to which they can help prevent oxidative

46 stress and lifestyle diseases (Ficco et al., 2014; Giordano et al., 2017; Luthria, Lu, & John,

47 2015).

48 A wide range of carotenoids may be present in wheat grain including lutein, -

49 carotene, -cryptoxanthin, zeaxanthin, antheraxanthin, taraxanthin (lutein 5,6-epoxide),

50 triticoxanthin, and flavoxanthin (Barnes, 2012). In general, lutein is the most abundant

51 carotenoid, followed by zeaxanthin, antheraxanthin, -carotene, and -carotene, while

52 -cryptoxanthin is a minor component or it occurs at non-detectable levels (Adom, Sorrells, 

53 Liu, 2003; Paznocht et al., 2018). Zeaxanthin in wheat is interconverted to the mono-

54 epoxidated xanthophyll antheraxanthin, but the further product of the xanthophyll cycle,

55 violaxanthin, has not been detected in wheat grain (Paznocht et al., 2018). Total carotenoid

56 content (TCC) in wheat falls in the range of 3.0–10.2 g/g DW depending on wheat species

57 (Hidalgo, Fongaro, & Brandolini, 2017). These authors reported the highest carotenoid
 4

58 content in einkorn wheat (Triticum monococcum ssp. monococcum), followed in descending

59 order by Polish wheat (Triticum turgidum ssp. polonicum), Khorasan wheat (Triticum

60 turgidum ssp. turanicum), durum wheat (Triticum turgidum ssp. durum), spelt (Triticum

61 aestivum ssp. spelta), bread wheat (Triticum aestivum ssp. aestivum), and emmer wheat

62 (Triticum turgidum ssp. dicoccum).

63 Colored-grain wheat genotypes, which are currently an object of interest to breeders

64 and consumers, can in addition to anthocyanin pigments, also contain meaningful (not

65 negligible) amounts of carotenoids. A recent study (Paznocht et al., 2018) deals with the

66 content and composition of carotenoids in unusual colored wheat varieties. They found that

67 blue wheat genotypes had lower TCC, whereas purple ones had the same or higher levels of

68 carotenoids than conventional bread wheat.

69 Carotenoids and anthocyanins are natural pigments that degrade due to both external

70 factors (e.g., high temperature, extreme pH values, oxygen presence, UV light) and internal

71 factors (e.g., enzymatic activity) (Leenhardt et al., 2006). The characteristic highly

72 unsaturated structure of carotenoids is the source of their beneficial effects but also of their

73 particular instability, especially toward light and oxygen. Carotenoid degradation is a complex

74 process involving many factors linked to the food matrix and the type of carotenoid (Achir,

75 Randrianatoandro, Bohuon, Laffargue, & Avallone, 2010). Carotenoids occur in foods

76 predominantly as E-isomers, though Z-isomers are formed during thermal processing and

77 show different biological properties, bioavailability, and antioxidant activity. In cereals,

78 carotenoids are present either in free or esterified forms (mostly with palmitic and linoleic

79 acid) depending on the cereal genotype (Ziegler, Wahl, Würschum, Longin, Carle, &

80 Schweiggert, 2015). It has been shown that esterified forms of xanthophylls are more stable in

81 the presence of heat than their free forms (Subagio, Wakaki, & Morita, 1999). The potential

82 health benefits of these susceptible substances is therefore influenced by grain processing

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83 (from milling to final technological processing), which can significantly reduce the amount of

84 the nutritionally valuable compounds in foodstuffs (Hidalgo, Brandolini, & Pompei, 2010).

85 The effect of processing (boiling, baking, etc.) on the content of carotenoids and

86 anthocyanins has been discussed in many studies. Some of these describe the degradation of

87 carotenoids or anthocyanins in cereal grains and consequent cereal, baked, or puffed products

88 (Abdel-Aal, & Hucl, 2014; Hidalgo et al., 2010; Hidalgo, Scuppa, & Brandolini, 2016).

89 Currently, newly bred genotypes of colored-grain cereals seem to be potentially valuable

90 sources of these phytochemicals in the human diet (Paznocht et al., 2018).

91 Therefore, the aim of this study was to compare carotenoid content and profile in differently

92 colored wheats and their products. We focused on colored wheat because it is a novel and

93 apparently healthier alternative to conventional wheat. Keeping in mind the important health

94 benefits of carotenoids and high per capita wheat consumption, the objectives of this study

95 were: i) to establish the carotenoid profile and content in flour from each genotype; ii) to

96 investigate the changes in carotenoids in dough prepared from colored-grain wheat flours; iii)

97 to establish the changes of carotenoid content in buns after baking and after a short 24-hour

98 storage.

99

100 2. Material and methods

101 2.1. Plant material

102 A total of nine colored-grain wheat varieties and breeding materials were grown in

103 2016/2017 at the Agricultural Research Institute in Kroměříž, Czech Republic (49.2851172N,

104 17.3646269E). The experimental field is located 235 meters above sea level, has Luvic

105 Chernozem (Loamic) soil, an average annual temperature 9.2 °C, mild winters, and annual

106 precipitation averaging 576 mm. The plants were grown on small experimental plots (10 m2)

107 using conventional growing technology. Samples were stored after the harvest in paper bags
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108 in a box in the dark at room temperature of 21 °C for 2 months before being analyzed. The

109 main characteristics of the selected wheat genotypes are listed in Table 1.

110 2.2. Preparation of wheat flour, dough, baking of buns and their storage after baking

111 2.2.1. Preparation of wholemeal wheat flour

112 The grains were cleaned on a Labofix (C.W. Brabender Instruments, Inc.; South

113 Hackensack, NJ) over a 2-mm sieve, and impurities were removed manually. The grain was

114 milled on a YM1 Wet Wheat Grinding Machine Y-10 (Yucebas Machine Analytical

115 Equipment Industry, Izmir, Turkey), and three fractions were obtained in total: coarse bran

116 (>35 mesh), fine bran (50–35 mesh), and flour (<50 mesh). All the fractions were mixed

117 together resulting in total recovery of the grain in so-called wholemeal flour.

118 2.2.2. Preparation of dough, baking, and short-term storage of baked buns for 24 h

119 For the preparation of the doughs, 300 g wholemeal flour, 4.8 g table salt, 6 g

120 compressed commercial baker’s yeast, and 180 mL warm (30 C) water were used. The

121 dough-making process was as follows: 30 min kneading, 1 h rising, and short kneading (2

122 min) in the middle of the rising period (Alaska BM 2000; SIG GmbH, Düsseldorf, Germany).

123 After rising, the dough was divided into 3 buns. Two of them were baked at 240 °C for 14

124 minutes in a forced air oven Venticell 111 (BMT Medical Technology, Ltd., Brno, Czech

125 Republic); the oven also contained a water bath containing 70 mL of distilled water. After

126 baking, one bun was frozen immediately after cooling and the other after 24-h storage at room

127 temperature (21 °C) under a cloth. The unbaked third bun was frozen immediately after

128 separation from the other parts of dough. All samples were then freeze-dried (Lyovac GT2;

129 Steris, Hürth, Germany) in the dark for 120 h and then subjected to further analyses. The bun

130 experiments were performed in three replicates per genotype.

131

132
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133 2.3. Chromatographic analysis of carotenoids

134 2.3.1. Chemicals

135 2.3.2.

136

137 Chemicals

138 Lutein and zeaxanthin standards (UV, ≥ 95%, ≥ 98%) were obtained from

139 Extrasynthèse, Genay, France. β-Carotene standard (HPLC, ≥ 95%), ethanol absolute (puriss.,

140 ≥ 99.8%), butylated hydroxytoluene (BHT, ≥ 99% FG), and tert-butyl methyl ether (HPLC

141 grade) were purchased from Sigma-Aldrich, St. Louis, MO. Antheraxanthin and α-carotene

142 standards (HPLC, 95%, 97%) were purchased from CaroteNature, GmbH, Lupsingen,

143 Switzerland. Methanol (HPLC grade), acetone (GR grade), ethanol (GR grade), and hexane

144 (GR grade) were purchased from Lachner Ltd., Neratovice, Czech Republic. Ultra-pure

145 HPLC water was prepared using Simplicity UV (Merck Millipore, KGaA, Darmstadt,

146 Germany).

147 2.3.2. Sample extraction

148 Carotenoids were extracted according to the method modified for the grain matrix

149 (Kotíková, Šulc, Lachman, Pivec, Orsák,  Hamouz, 2016). Finely ground sample (2 g) was

150 placed into a 50-mL plastic Falcon tube with 12 mL ethanol/acetone/hexane mixture (1:1:2,

151 v/v/v), vortexed, and left to stand for 24 h in a refrigerator (4 °C). The sample was then

152 vortexed for 1 min (Basic 3; IKA Werke GmbH & Co. KG, Staufen, Germany), sonicated for

153 10 min in an ultrasonic bath (PS 04; Powersonic-Notus, Ltd., Vráble, Slovakia), and

154 centrifuged at 8228 rcf for 10 min (5810R; Eppendorf, Hamburg, Germany). Then 9 mL of

155 the supernatant were transferred into a 50-mL glass evaporation flask and the sediment re-

156 extracted with 12 mL of the extraction mixture. Both supernatants were combined and

157 evaporated under vacuum at 40 °C (Rotavapor R-200; Büchi Labortechnik, AG, Flawil,
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158 Switzerland). The dry residue was reconstituted with 2 mL ethanol/acetone (3:2, v/v)

159 containing 0.2% BHT and filtered through a syringe filter (PVDF, 0.45 μm) into an amber

160 HPLC vial.

161

162 2.3.3. Chromatographic separation by HPLC-DAD

163 Analyses were carried out using an Ultimate 3000 HPLC system (Thermo Fisher

164 Scientific, Waltham, MA) with a quaternary pump, autosampler, column heater, and diode

165 array detector. The analytes were separated by gradient elution on an YMC C30 Carotenoid

166 Column (150 mm × 3.0 mm, S-3 μm; YMC Co., Kyoto, Japan). The operating conditions

167 were as follows: flow rate 0.6 mL/min; column temperature 25 °C; autosampler temperature

168 10 °C; injection volume 10 μL; detection at λ = 445 nm (spectral acquisition 300–700 nm).

169 The tertiary mobile phase consisted of methanol (A), water (B), and tert-butyl methyl ether

170 (C). Gradient: initial conditions of 90% A, 10% B, and 0% C were sustained for 1 min, then

171 increased to 90% A, 0% B, and 10% C at 6 min, reaching 40% A, 0% B, and 60% C at

172 22 min following column flush and re-equilibration for 11 min.

173 2.3.4. Identification and quantification

174 The carotenoids were identified by comparing retention times and absorption spectra

175 with those of analytical standards. The quantification of free forms was based on peak area

176 and external calibration (concentration range 0.01–10 μg/mL per analyte). Analyses were

177 performed in triplicate. The exact concentration of carotenoid stock solutions was determined

178 spectrophotometrically (Spectronic Helios γ; Thermo Fisher Scientific, Waltham, MA) using

179 the following extinction coefficients (EC, L/g/cm): antheraxanthin (235, λmax = 446 nm),

180 lutein (255, λmax = 445 nm), both in ethanol; zeaxanthin (234, λmax = 452 nm) in acetone; β-

181 carotene (259.2, λmax = 453 nm) and α-carotene (271, λmax = 445 nm), both in hexane. The

182 presence of lutein esters was confirmed by alkaline hydrolysis of the sample extract (Paznocht
 9

183 et al., 2018). Since esterification with fatty acids does not affect chromophore properties

184 (Mellado-Ortega & Hornero-Méndez, 2017), lutein esters were quantified using the all-E-

185 lutein calibration curve. Since most of the esters in wheat are lutein esters with only a minor

186 proportion of antheraxanthin and zeaxanthin esters (Paznocht et al., 2018), we have expressed

187 them in this article summarily as lutein esters. The Z-isomers of lutein were confirmed by

188 photoisomerization of lutein by iodine. Particular Z-forms were identified by exploring their

189 absorption spectra (Paznocht et al., 2018). Their quantification was based on the calibration

190 curve of all-E-lutein. Limits of detection (LOD) for antheraxanthin, lutein, zeaxanthin, α-

191 carotene, and -carotene (0.004, 0.006, 0.012, 0.010, and 0.015 g/g DW, respectively) were

192 calculated using the formula 3.3 × (σ/S) (Q2B CH, 1996). An illustrative chromatogram of the

193 Bona Vita variety (yellow endosperm) showing differences between flour, dough, bun and

194 bun stored for 24 h is shown in Fig. 1.

195 2.3.5. Dry weight (DW)

196 Dry weight (calculated as loss of water) was determined by drying 10 g of wholemeal

197 flour at 105 °C for 24 h in the forced-air oven Venticell 111.

198 2.4. Statistical analysis

199 The data were processed by Chromeleon (Thermo Fisher Scientific, Inc., Waltham,

200 MA) and Excel (Microsoft, Redmond, WA). Statistical evaluation was performed using

201 STATISTICA software (ver. 12; StatSoft, Inc., Tulsa, OK). The effects of variety and

202 technological processes on the carotenoid content were evaluated by one-way and two-way

203 ANOVA (p ≤ 0.05). Tukey's Post Hoc HSD test was used for detailed statistical evaluation.

204

205 1. Results and discussion Commented [S1]:

Commented [S2]: Check heading numbering throughout
206 The profiles of individual carotenoids in flour, dough, baked buns, and buns stored for 24 document.

207 h after baking are reported in Table 2.

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208 3.1. Carotenoid content and profile in wheat flour

209 The average total carotenoid content (TCC) in wholemeal flour in all analyzed

210 genotypes was 1.05 µg/g DW and ranged from 0.378 ± 0.004 μg/g DW (var. Bohemia) to

211 2.24 ± 0.044 µg/g DW (var. Citrus). The highest TCC was observed in winter yellow-grained

212 Citrus and Bona Vita flours. Elevated contents were also found in flour from purple spring

213 wheat Konini and purple winter wheat AF Jumiko. TCC in flour from varieties and breeding

214 materials with blue aleurone (V1 131-15 and UC 66049) and red-grained control varieties

215 (Annie and Bohemia) was relatively low.

216 Wholemeal flour is composed of 10–14% bran, 2.5–3.0% germ and 80–85%

217 endosperm. The bioactive compounds are distributed within these parts and are concentrated

218 in the outer layer of the grain. Thus, wholemeal flour contains substantially more

219 phytonutrients, including carotenoids, than refined wheat flour (Mellado-Ortega & Hornero-

220 Méndez, 2016). Our TCC values for wholemeal flour originating from conventional winter

221 Annie and Bohemia varieties were on average 23% higher than those measured by Lv et al.

222 (2012) in soft red winter wheat flours and almost twice as high as the values for refined flours

223 reported by Konopka, Czaplicki, and Rotkiewicz (2006). Similarly, Ficco et al. (2016), who

224 used different types of conventional and purple durum wheat flours to produce fresh pasta,

225 found the highest TCC in wholemeal flour (7.38 g/g DW), followed by semi-wholemeal

226 (6.30 g/g DW), and the lowest in semolina (5.25 g/g DW). They reported that TCC in

227 purple durum wheat (7.38 g/g DW) was slightly lower in comparison with yellow durum

228 wheat wholemeal flour (7.68 g/g DW). However, some of our pigmented varieties,

229 especially purple ones, reached comparable or even higher values of TCC than conventional

230 red varieties. Ndolo and Beta (2013) also found in their study higher TCC in wholegrain

231 fraction of purple wheat (2.62 g/g) than the average of three non-colored wheat varieties

232 (2.56 g/g). The authors found a more obvious difference between purple and non-pigmented
 11

233 barleys: TCC of purple barley (4.54 g/g) was twice as high as its non-pigmented form (2.25

234 g/g). These results, as well as the results of our previous study (Paznocht et al., 2018),

235 revealed that some purple pericarp (Pp) genotypes would be valuable sources of carotenoids

236 as well as anthocyanins, which are also important bioactive compounds in purple and blue

237 bread wheat varieties (Ficco et al., 2014). The increased content of carotenoids in purple

238 wheat grain could be explained by the genetic origin of the plants. Purple grain color is

239 conferred by the Pp genes, which were transferred to common wheat from tetraploid wheat

240 (Triticum turgidum L. subsp. abyssinicum Vavilov, genome AABB) originating from the East

241 Africa region (Lachman, Martinek, Kotíková, Orsák, & Šulc, 2017). According to older data,

242 the purple pericarp genes are located on chromosomes 7B (Pp1), 6A (Pp2), and 2A (Pp3a and

243 Pp3b) (Dobrovolskaya, Arbuzova, Lohwasser, Röder, & Börner, 2006). More recent studies

244 indicate that the purple pericarp is determined by complementary genes Pp3 (on 2A in bread

245 and durum wheat), Pp-B1 (on 7B in bread and durum wheat), or Pp-D (on chromosome 7D in

246 bread wheat) (Tereshchenko, Gordeeva, Arbuzova, Börner, & Khlestkina, 2012;

247 Tereshchenko, Pshenichnikova, Salina, & Khlestkina, 2012). Phytoene synthase enzymes are

248 responsible for the synthesis of yellow compounds, which are conditioned by genes located

249 on the long arms of the homoeologous group 7 chromosomes (7AL, 7BL, and 7DL) (Komugi

250 - Wheat Genetic Resources Database, 2017). In the AF Jumiko variety, the donor of purple

251 pericarp was ANK-28A, in which the anthocyanins were conditioned by the genes Pp1 (7B)

252 and Pp2 (6A) (Watanabe, Koval,  Koval, 2003). It is possible to theorize that there could be

253 a genetic link between the Pp1 gene and some of the genes of the Psy1-B1 locus (at 7BL) for

254 phytoene synthase enzymes. Because most of the studied donors of wheat with purple

255 pericarp carry the Pp1 gene and differ from each other in other Pp genes, it can be assumed a

256 relation between increased anthocyanins and an increased content of yellow carotenoids. This

257 hypothesis needs to be confirmed by other experiments.

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258 The composition of individual carotenoids in wholemeal flours of the analyzed

259 genotypes was very similar. The highest content was typical for total lutein (the sum of all-E-

260 lutein and other detected Z-isomers) (on average 0.695 µg/g DW), followed by its esters,

261 zeaxanthin, -carotene, -carotene, and antheraxanthin (0.244, 0.061, 0.033, 0.010, and 0.010

262 µg/g DW, respectively). The lutein fraction has been separated into all-E-lutein (0.615 µg/g

263 DW), followed by 13´-Z-lutein, 13-Z-lutein, 9-Z-lutein, and 9´-Z-lutein (0.031, 0.030, 0.014,

264 and 0.005 µg/g DW, respectively) in wholemeal flour on average in all analyzed genotypes.

265 While some carotenoids, such as all-E-lutein, its Z-isomers, and zeaxanthin, were detected in

266 all samples, others were detected only in some specific accessions, as in the case of

267 antheraxanthin, β-carotene, and α-carotene (Table 2). Total lutein content (TLC) correlated

268 positively with TCC. The highest TLC was found in yellow-grained Citrus and Bona Vita

269 varieties (1.85 and 1.03 g/g DW, respectively), followed by purple-grained varieties. Higher

270 TLC was also determined in the V1 131-15 genotype (Ba) where the yellow ancestors Citrus

271 and Bona Dea can contribute to higher TLC content (Skorpion × PS Karkulka) × (Citrus ×

272 Bona Dea). Lower TLC was observed in flours from blue-grained UC 66049 and standard

273 red-grained varieties.

274 The second most represented zeaxanthin ranged from 0.034 to 0.110 g/g DW with

275 the highest content in Konini (Pp) and V1 131-15 (Ba) flours and the lowest in Bona Vita

276 (Ye) flour. Antheraxanthin, α-carotene and β-carotene represented only minor components of

277 the carotenoid profile not exceeding 1.9% (var. Bohemia), 2.1% (var. Bona Vita) and 6.6%

278 (var. Bona Vita) of the TCC.

279 Giordano et al. (2017) compared the main carotenoids  lutein and zeaxanthin 

280 contained in wholemeal flour of red-, white-, yellow-, purple-, and blue-grained wheat

281 varieties. Also in their study, lutein was the major carotenoid followed by zeaxanthin. Similar

282 carotenoid profiles in wheat species have been identified by a number of other authors
 13

283 (Abdel-Aal, Young, Rabalski, Hucl,  Fregeau-Reid, 2007; Adom et al., 2003; Digesú,

284 Platani, Cattivelli, Mangini, & Blanco, 2009; Hidalgo et al., 2010; Hidalgo & Brandolini,

285 2014; Konopka et al., 2006). Most of these authors also identified minor amounts of β-

286 cryptoxanthin, which was not identified in our samples. On the other hand, none of the cited

287 authors found antheraxanthin across different wheat species. The absolute lutein

288 predominance in wheat grains observed in our samples, as well as in samples of all the above-

289 mentioned authors, is probably due to the increased activity of enzymes involved in the β,ε-

290 branch carotenoid biosynthesis. The carotenoid biosynthesis pathway is split from all-trans-

291 lycopene into two competing tracks. One track (β,ε-branch) leads to α-carotene and finally to

292 lutein, while the other (β,β-branch) leads to β-carotene and then to the oxygenated products -

293 cryptoxanthin, zeaxanthin, violaxanthin, and neoxanthin (Sun, Yuan, Cao, Yazdani, Tadmor,

294  Li, 2018). The higher activity of two specific β,ε-branch enzymes, lycopene ε-cyclase

295 (LCY-e) and heme-containing cytochrome P450 hydroxylase (CYP97C), catalyzing the

296 cyclization of all-trans-lycopene and the final hydroxylation of zeinoxanthin to lutein, can be

297 responsible for higher lutein content in wheat grains.

298 It follows from the results of our study that most of the investigated wheat genotypes

299 (with the exception of Bohemia, which contained only free xanthophylls) produced esterified

300 forms of xanthophylls, which in some cases represent a significant portion of total carotenoids

301 stored in wheat flour (Table 2). A high portion of esterified xanthophylls were contained in

302 Bona Vita (0.843 g/g DW, 43.8%), Konini (0.438 g/g DW, 30.7%), and Annie (0.263 g/g

303 DW, 38.6%) varieties. In our previous study (Paznocht et al., 2018), we found that esterified

304 xanthophylls are represented mainly by lutein esters, accompanied by minor amounts of

305 antheraxanthin and zeaxanthin esters. Based on this finding, we attributed xanthophyll esters

306 to lutein and expressed them simply as the sum of lutein esters.
 14

307 Xanthophyll esterification is a common method of sequestering carotenoids in plants

308 and it is presupposed to be an effective mechanism in increasing their accumulation (Atienza,

309 Ballesteros, Martín, & Hornero-Méndez, 2007). Some authors have observed that xanthophyll

310 esterification varies widely among and within wheat species and is subject to genetic control

311 (Ahmad et al., 2015; Ziegler et al., 2015). Therefore, the Bohemia variety (which contained

312 only free carotenoids) seems to have no genetic predisposition to synthesize xanthophyll

313 esters. Similarly, Ziegler et al. (2015) reported an absence of lutein esters in seven out of

314 fifteen investigated bread wheat T. aestivum genotypes, whereas in the remaining genotypes

315 lutein esters accounted for 22.7–38.3% of TLC. Einkorn genotypes contained lutein esters

316 from 4.4% to 58.3% of TLC. In contrast, no durum wheat varieties contained lutein esters.

317 Particularly interesting is the fact that higher TCC in cereals seems to have no direct influence

318 on the final portion of xanthophyll esters (Paznocht et al., 2018; Ziegler et al., 2015).

319 3.2. Degradation of carotenoids during dough preparation

320 Results of the degradation of TCC, TLC, and other carotenoids during dough

321 preparation are given in Table 2. Percentage losses of TCC and TLC in individual varieties

322 and breeding materials during baking production are shown in Fig. 2. In the doughs, TCC in

323 all analyzed wheat genotypes decreased to 38.5% on average compared to its original content

324 in wholemeal flour. The preparation of dough was the most devastating step regarding the

325 degradation of carotenoids. The largest decreases in TCC during dough preparation were

326 recorded in the yellow wheat varieties Citrus and Bona Vita (to 24.9% and 25.7%,

327 respectively). In Pp genotypes, various losses were observed among the individual accessions.

328 The most considerable of these decreases occurred in the Konini variety (to 20.3%), which

329 was characterized by the highest beginning TCC among Pp genotypes. In other Pp genotypes,

330 moderate losses in TCC were recorded: in AF Jumiko, TCC decreased to 56.9% and in ANK-

331 28A to 64.7% of the original content in wholemeal flour. A similar situation was observed in
 15

332 Ba genotypes. A significant decrease in TCC was recorded in the UC 66049 genotype (to

333 36.3%), while in the V1 131-15 genotype, carotenoid losses were significantly smaller (to

334 85.0%). Conventional wheat varieties were also characterized by different carotenoid losses

335 during dough preparation; in variety Bohemia TCC decreased to 31.7%, and in variety Annie

336 to 74.5% (Fig. 2).

337 Among individual carotenoids, the highest losses were recorded for

338 α-carotene and β-carotene. Originally contained in the flours only in trace amounts, α-

339 carotene totally decomposed and was not detected in doughs (Table 2). However, not a total

340 loss, β-carotene content dropped to 34.2% of its initial value in flour. A significant decrease

341 also occurred in TLC (to 35.8%). Lutein esters appeared to be more stable compared to free

342 lutein, as evidenced by their smaller decrease (to 43.9%). Zeaxanthin and antheraxanthin were

343 evaluated as the carotenoids most resistant to the dough preparation process, with the lowest

344 reduction in their content (to 54.0% and 53.6%, respectively). Focusing on lutein forms, the

345 major all-E-lutein decreased to a considerable extent (to 33.3%) of the original content. In

346 particular, the behavior of Z-isomers was different during the preparation of dough. The

347 content of 13-Z-lutein dropped even more than its all-E counterpart (to 26.4%), whereas

348 losses in other Z-isomers were significantly lower. 13´-Z-Lutein and 9-Z-lutein decreased

349 moderately (to 63.5% and 80.5%, respectively), and the decrease of the least-contained 9´-Z-

350 lutein was only negligible (to 98.3%). These results suggest an enhanced stability of certain Z-

351 isomers of lutein and/or ongoing isomerization of all-E-lutein to its particular Z-counterparts

352 during the dough preparation process.

353 Carotenoids are susceptible to a number of exogenous factors, such as light, oxygen,

354 heat, and extreme pH values, as well as to endogenous enzymatic activity. Their losses during

355 dough preparation take place due to the presence of oxidative enzymes, such as lipoxygenase,

356 peroxidase, and polyphenol oxidase, in the flour, which become active when water is added
 16

357 and oxygen is incorporated during the process (Ficco et al., 2016; Leenhardt et al., 2006;

358 Luthria et al., 2015). In our study, TCC decreased by 61.5% during dough preparation, which

359 is comparable to the significant decrease in TCC (by 66.0%) of bread wheat wholemeal flour

360 during kneading previously reported by Leenhardt et al. (2006). Fratianni, Di Criscio,

361 Mignogna, and Panfili (2012) also reported significant reduction in TCC (by 48.9%) in dough

362 during egg pasta-making process. The above cited authors found high correlation between

363 carotenoid losses and the lipoxygenase (LOX) activity, while peroxidase was far less active

364 and had little impact on carotenoid degradation and related dough bleaching (Leenhardt et al.,

365 2006). LOX catalyzes the oxidation of polyunsaturated fatty acids, which leads to the

366 subsequent oxidation of carotenoid pigments. LOX activity is highly dependent on wheat

367 species (Hidalgo et al., 2010; Hidalgo & Brandolini, 2012; Leenhardt et al., 2006). Hidalgo

368 and Brandolini (2012) reported the highest lipoxygenase activity in flour of T. aestivum (8.02

369 µmol/min/g DW), followed by T. turgidum (3.48 µmol/min/g DW) and T. monococcum (0.45

370 µmol/min/g DW). Similarly, Hidalgo et al. (2010) observed different damage levels between

371 wheat species during kneading associated with their different LOX activity (in descending

372 order, bread wheat, durum wheat, and einkorn). These results indicate a high lipoxygenase

373 activity in bread wheat and lower activity in high-carotenoid varieties. In our high-carotenoid

374 varieties Citrus, Bona Vita, and Konini, there were significant decreases in TCC, probably

375 because they are T. aestivum varieties. Other important factors influencing LOX activity are

376 the time, intensity, and temperature of dough kneading (Hidalgo et al., 2010), the pH value of

377 the dough  enzymatic activity of LOX is highest over the pH range 5–6 (Hidalgo, 

378 Brandolini, 2012)  and the percentage and composition of lipids in the grain. Higher

379 proportions of unsaturated fatty acids promote the production of peroxides and other free

380 radicals that can react with the carotenoid compounds (Cueto, Farroni, Schoenlechner,

381 Schleining, & Buera, 2017). Therefore, to improve carotenoid stability during the dough
 17

382 preparation process, it is crucial to choose appropriate wheat species and varieties with low

383 LOX activity and appropriate lipid composition and to minimize the time and intensity of

384 kneading.

385

386

387 3.3. Degradation of carotenoids during heat treatment (baking) and short-term bun

388 storage

389 Heat treatment during bun baking caused further carotenoid degradation (by 11.1%),

390 decreasing TCC to 27.4% of the original content in flour. This means that baking led to

391 a substantially smaller reduction of carotenoids as compared to dough preparation (Table 2;

392 Fig. 2). The most significant relative decreases of TCC were recorded in the red wheat Annie

393 (by nearly one-third, to 44.0%) and the Pp wheat ANK-28A (by nearly one-fourth, to 40.5%).

394 However, the least significant TCC decreases were observed in the red wheat Bohemia and

395 yellow-grained wheat Citrus (by 2.1% and 5.0%, respectively). In buns formed with the Pp

396 wheat Konini, a slight increase (of 2.2%, to 22.5%) was recorded. Significantly higher TCC

397 reduction (by 29%) occurred in the bread crust during baking, but carotenoids present in the

398 bread crumb were substantially more stable (experiencing a decrease of only 3%) as reported

399 by Hidalgo et al. (2010). According to these authors, heat treatment had a major effect on

400 overall TCC.

401 Total lutein decreased by 10.6% (Table 2, Fig. 3). All-E-lutein remained the major

402 carotenoid despite its amount falling by 9.7%. Considerable decrease of approximately one-

403 third was found for 13´-Z-lutein (by 29.1%). In the case of two lutein Z-isomers, 9-Z- and 9´-

404 Z-lutein, their amounts were reduced by around one-quarter and one-half, respectively, though

405 despite these substantial losses, 62.4% and 47.7% of the original values remained. Hence, we

406 assume a higher stability of 9-Z- and 9´-Z-lutein in comparison to other lutein isomers and/or
 18

407 other reactions resulting in isomerization from E- to certain Z-isomers. Baking caused

408 significant decreases of lutein esters (by 22.2%), β-carotene and antheraxanthin (by 23.8%

409 and 30.8%, respectively). On the other hand, an increase was recorded in individual

410 carotenoids such as zeaxanthin, which increased to 89% (an increase of 35%) of its original

411 content in flour and 13-Z-lutein, which increased to 28.4% (an increase of 2%). It is

412 understood that thermal processing induces carotenoid isomerization of E-isomers to

413 particular Z-forms (Cueto et al., 2017; Updike, & Schwartz, 2003). Hidalgo et al. (2010)

414 further hypothesize the hydroxylation of carotenes took place caused by high temperatures,

415 due to the significant increase of β-cryptoxanthin content in bread crust observed in their

416 experiment.

417 After the short-term storage of buns for 24 h, only a small additional decrease in TCC

418 was observed (reaching 24.9% of wholemeal flour, a decrease of 2.5%). The largest storage-

419 related losses were found in wheats V1 131-15 and Annie (by 10.8% and 9.2%, respectively).

420 Regardless, in buns made of wheat V1 131-15 (blue, crosses with Citrus ancestor), the highest

421 TCC remained at the end of the baking and storage process (53.1%). However, the V1 131-15

422 grain buns retained their relatively highest TCC after the production and storage processes.

423 From the viewpoint of the final TCC measurement in our study, this genotype occupies

424 second place behind the yellow-grained wheat Citrus (0.431 g/g DW). The high TCC

425 preserved in buns after storage was mainly due to the high carotenoid content in the original

426 flour. The mildest fall of TCC was observed in wheat Citrus (a decrease of 0.6%). Two

427 samples of Pp wheat (ANK-28A and AF Jumiko) showed no decrease or only a very slight

428 increase (of 0.5% and 2.7%, respectively).

429 Similarly, concentrations of individual analytes did not change significantly during the

430 24 h of storage. The major carotenoid lutein dropped by only 2.2%. The largest decreases

431 were observed in zeaxanthin (a drop of 11.4%) and 9-Z-lutein (a drop of 10.7%) levels. On
 19

432 the contrary, 9´-Z-lutein increased by 18.6%. Decline of TCC during short-term storage was

433 probably caused by the presence of atmospheric oxygen (Hidalgo et al., 2010).

434 In total, during the full process from flour to final product, carotenoids degraded by

435 75.1% (i.e., 24.9% of TCC was preserved). Distinctly higher carotenoid losses (92%) in wheat

436 (Triticum aestivum) bread have been reported by Leenhardt et al. (2006). Hidalgo et al. (2010)

437 reported TCC decrease in bread made of Blasco wheat (Triticum aestivum) by 24% and 55%

438 in bread crumb and crust, respectively. The differences between obtained results may be

439 caused by using different bread-making protocols.

440 In our study, the highest degradation was characteristic of -carotene and the most

441 resistant to decomposition was zeaxanthin (Fig. 3), likely due to possible isomerization of

442 lutein to zeaxanthin or increased hydroxylation of carotenes at higher temperatures (Hidalgo

443 et al., 2010). In the present study, bound esterified carotenoids degraded to a similar extent as

444 free carotenoids, and there was not observed any significant protective effect of carotenoid

445 esterification on stabilization and resistance to heat thermal treatment, as was reported

446 previously in some studies (Ahmad et al., 2015; Subagio et al., 1999). Adding a layer of

447 complexity, Z-isomers of lutein in our samples showed a lesser decrease in final buns

448 compared with all-E-lutein. This can be explained by the isomerization of E-forms to Z-forms

449 through thermal processing, as has also been observed in vegetables (Updike, & Schwartz,

450 2003).

451 Although the results of two-way statistical analysis of variance ANOVA using Tukey's

452 HSD test (data is not shown) revealed slightly lower average TCC losses in purple- and blue-

453 grained wheat (25.7% and 27.2%, respectively) as compared with red-grained conventional

454 control wheat (28.7%) and higher losses in yellow-grained wheat (39.3%), the differences

455 between them were statistically not significant (p > 0.05). That is, the stability of carotenoids
 20

456 in relation to the color of the grain showed only a weak tendency (in the presence of

457 anthocyanins), but were highly dependent on a particular genotype.

458 Conclusion

459 In conclusion, the genotypes of new colored-grain wheat varieties, lines, and breeding

460 materials, as well as the various factors of the preparation, storage, and baking processes may

461 significantly affect the carotenoid levels and other health components in wheat flour and final

462 baked products. These results may serve as a background for wheat breeding efforts to

463 produce wheat flours and bakery products beneficial to human health. Therefore, it may be

464 possible for wheat breeders to select genotypes that optimize the levels of selected health

465 components. Future investigations should examine two paths: first, breeding approaches that

466 can be used to select high-carotenoid and high-anthocyanin cultivars with low activity of

467 oxidative enzymes and appropriate composition of grain lipids; and second, new

468 technologically optimized approaches, like air-classification, vacuum dough making, and

469 optimal durations of kneading, baking, heat treatment, extrusion, and puffing.

470 Conflicts of interest

471 The authors have declared no conflicts of interest regarding this work.

472 Acknowledgments

473 This work was supported by the project NAZV No. QJ1510206 of the Ministry of

474 Agriculture of the Czech Republic and CIGA CULS project No. 20182004.

475 We would like to thank Matthew T. Jones for his thorough proofreading of this article.

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590 TABLE AND FIGURE CAPTIONS

591 Figure 1. Chromatogram of Bona Vita wheat variety (Ye) showing differences between flour

592 (A), dough (B), bun (C), and bun after 24 h of storage (D).

593 1 – (13-Z)-Lutein (9.89 min), 2 – Antheraxanthin (10.00 min), 3 – (13-´Z)-Lutein (10.20 min),

594 4 – All-E-Lutein (10.70 min), 5 – Zeaxanthin (11.35 min), 6 – (9-Z)-Lutein (11.53 min),

595 7 – (9´-Z)-Lutein (12.24 min), 8 – α-Carotene (15.30 min), 9 – β-Carotene (16.25 min),

596 10 – Lutein monoesters, 11 – Lutein diesters.

597 Figure 2. Percentage loss of total carotenoid content (TCC) during baking production for

598 each variety and genotype (%).

 26

599 Figure 3. Evolution of total lutein and zeaxanthin content on average of all analyzed varieties

600 and genotypes during baking production (%).

601 Table 1. Description of bread wheat (Triticum aestivum L.) varieties and genotypes.

602 (1) CZE – Czech Republic, DEU – Germany, SVK – Slovak Republic, NZL– New Zealand,

603 RUS – Russian Federation, USA – United States of America

604 (2) V1 131-15: (Skorpion × PS Karkulka) × (Citrus × Bona Dea)

605 Table 2. Content of carotenoids  STD (µg/g DW) in wheat flour, dough, bun after baking,

606 and bun after 24 h of storage and their decrease (%) as compared to the content of wholemeal

607 flour.

608 n.d. – not detectable; STD  standard deviation; values in columns marked with different

609 letters are statistically different at p  0.05; *average of all analyzed varieties and genotypes

610 Figure 1.

611

612

mAU WVL: 445 nm

4 var. Bona Vita (Ye)
40

30

10 11

20
8 9
7
12 3 5 6
A
10
B

C
0
D
min

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

613

614

615
 27

616

617

618

619

620 Figure 2.

621

622 Bohemia Annie

100.0%
Total carotenoids [µg/g DW]

Total carotenoids [µg/g DW]

623 0.5 0.8 100.0%

0.4 0.6 74.5%

624 Figure
0.3 3.
0.4 44.0%
31.7% 29.6% 34.8%
0.2 25.5%
625 0.1
0.2
1.40 0.09 100.0% 89.0%
0.0 0.0
626 100.0% flour dough bun bun after 24 h
flour dough bun bun after 24 h 0.08 77.6%
1.20
0.07
DW]

Zeaxanthin [µg/g DW]

627 1.00 Citrus Bona Vita

0.06
[µg/g

100.0% 54.0%
Total carotenoids [µg/g DW]

0.80
[µg/g DW]

2.5 100.0% 0.05

628 2.5
Total lutein

2.0 0.04
2.0
0.60
629 1.5
Total carotenoids

35.8% 1.5
0.03
0.40
1.0 24.9%
25.2% 23.0% 1.0
19.9% 19.3% 0.02 25.7%
630 0.5 0.5 12.9% 11.1%
0.20
0.01
0.0 0.0
631 0.00 flour dough bun bun after 24 h 0.00 flour dough bun bun after 24 h

flour dough bun bun after flour dough bun bun after
632 24 h 24 h
V1 131-15 UC 66049
Total carotenoids [µg/g DW]
Total carotenoids [µg/g DW]

100.0%
633 0.8 0.5 100.0%
85.0%
0.4
0.6 63.9%
634 53.1% 0.3
0.4
0.2 36.3%
23.6% 21.9%
635 0.2 0.1
0.0 0.0
flour dough bun bun after 24 h flour dough bun bun after 24 h
636

637
AF Jumiko Konini
Total carotenoids [µg/g DW]
Total carotenoids [µg/g DW]

1.0 1.8
Table 1. Description
100.0% of bread wheat (Triticum aestivum L.) varieties
100.0% and genotypes.
0.8 1.5
1.2
Variety/
0.6 Growth 56.9%Country of origin
41.4% Variety status Grain color
38.7%
0.9
genotype
0.4 type (1) 0.6 20.3% 22.5% 20.1%
Bohemia
0.2 Winter CZE Released0.3
variety Red (standard)
Annie 0.0 Winter doughCZE bun
flour
Released varietyflour
bun after 24 h
0.0 Red (standard)
dough bun bun after 24 h

ANK-28A
µg/g DW]

0.8 100.0%

0.6 64.7%
 28

Yellow
Citrus Winter DEU Released variety
endosperm
Yellow
Bona Vita Winter SVK Released variety
endosperm
AF Jumiko Winter CZE Released variety Purple pericarp
V1 131-15
Winter CZE Breeding line Blue aleurone
(2)
UC 66049 Spring USA Genetic resource Blue aleurone
Konini Spring NZL Research germplasm Purple pericarp
ANK-28A Spring RUS Research germplasm Purple pericarp
638 (1) CZE – Czech Republic, DEU – Germany, SVK – Slovak Republic, NZL– New Zealand,
639 RUS – Russian Federation, USA – United States of America
640 (2) V1 131-15: (Skorpion × PS Karkulka) × (Citrus × Bona Dea)
641

642

643 Table 2. Content of carotenoids  STD (µg/g DW) in wheat flour, dough, bun after baking,
644 and bun after 24 h of storage and their decrease (%) as compared to the content of wholemeal
645 flour.
al step Antheraxanthin all-E-Lutein 13-Z-Lutein 13´-Z-Lutein 9-Z-Lutein 9´-Z-Lutein Zeaxanthin -Carotene -Carotene Esters Total lute
0.007±0.002a 0.260±0.002a 0.014±0.000a 0.013±0.000a 0.013±0.001a 0.011±0.001a 0.060±0.004a n.d. n.d. n.d. 0.312±0.0
n.d. 0.081±0.005b n.d. 0.008±0.001b 0.007±0.003b n.d. 0.024±0.002c n.d. n.d. n.d. 0.096±0.0
n.d. 0.060±0.003c n.d. 0.006±0.001b 0.006±0.002b n.d. 0.040±0.003b n.d. n.d. n.d. 0.072±0.0
h n.d. 0.055±0.000c n.d. 0.006±0.003b n.d. n.d. 0.035±0.003b n.d. n.d. n.d. 0.061±0.0
0.005±0.001a 0.320±0.010a 0.015±0.002a 0.016±0.000a 0.010±0.001a n.d. 0.054±0.003ab n.d. n.d. 0.263±0.009a 0.361±0.0
0.004±0.001a 0.232±0.009b 0.014±0.003a 0.018±0.004a 0.009±0.002a 0.008±0.000a 0.050±0.003bc n.d. n.d. 0.173±0.006b 0.281±0.0
n.d. 0.133±0.006c 0.011±0.003a 0.010±0.001b 0.007±0.001a n.d. 0.059±0.002a n.d. n.d. 0.080±0.004c 0.161±0.0
h n.d. 0.104±0.002d 0.008±0.004a 0.010±0.001b 0.007±0.002a n.d. 0.042±0.004c n.d. n.d. 0.066±0.003c 0.129±0.0
0.038±0.001a 1.637±0.032a 0.094±0.005a 0.074±0.002a 0.037±0.005a 0.012±0.002a 0.074±0.011a 0.025±0.002a 0.053±0.011a 0.194±0.012a 1.854±0.0
0.015±0.001b 0.343±0.013b 0.022±0.001b 0.020±0.004b 0.017±0.002b 0.008±0.002a 0.038±0.008b n.d. 0.025±0.002b 0.068±0.004b 0.411±0.0
0.009±0.000c 0.255±0.008c 0.023±0.001b 0.019±0.003b 0.018±0.003b 0.011±0.003a 0.065±0.011a n.d. n.d. 0.047±0.002c 0.325±0.0
h 0.008±0.001c 0.238±0.009c 0.019±0.002b 0.016±0.001b 0.014±0.000b 0.010±0.000a 0.063±0.004a n.d. 0.017±0.003b 0.046±0.004c 0.297±0.0
0.018±0.003a 0.915±0.016a 0.046±0.002a 0.044±0.003a 0.019±0.001a 0.007±0.001a 0.034±0.003a 0.044±0.010a 0.140±0.003a 0.843±0.045a 1.030±0.0
0.007±0.001b 0.157±0.010b 0.012±0.000b 0.016±0.004b 0.007±0.000b 0.008±0.001a 0.024±0.002b n.d. 0.047±0.011b 0.264±0.007b 0.200±0.0
n.d. 0.093±0.001c 0.011±0.002b 0.007±0.001c 0.007±0.001b n.d. 0.027±0.001ab n.d. 0.016±0.002c 0.109±0.001c 0.119±0.0
h n.d. 0.084±0.003c 0.009±0.003b 0.008±0.001c 0.007±0.002b n.d. 0.019±0.005b n.d. 0.017±0.000c 0.089±0.005c 0.109±0.0
n.d. 0.491±0.021a 0.015±0.001a 0.034±0.001b 0.014±0.002b n.d. 0.078±0.009b n.d. 0.020±0.003a 0.081±0.005a 0.554±0.0
0.006±0.000a 0.361±0.003b 0.012±0.002ab 0.044±0.003a 0.028±0.006a 0.014±0.003a 0.054±0.003c n.d. 0.015±0.002a 0.090±0.003a 0.459±0.0
n.d. 0.239±0.007c 0.011±0.001bc 0.022±0.001c 0.015±0.004b 0.009±0.001b 0.111±0.011a n.d. n.d. 0.063±0.002b 0.295±0.0
h n.d. 0.203±0.002d 0.008±0.001c 0.022±0.002c 0.012±0.004b 0.010±0.002ab 0.087±0.001b n.d. n.d. 0.048±0.004c 0.255±0.0
0.005±0.000a 0.196±0.006a 0.007±0.000a 0.014±0.001a n.d. n.d. 0.035±0.007a n.d. n.d. 0.144±0.006a 0.217±0.0
n.d. 0.056±0.002b n.d. 0.010±0.002b 0.006±0.003a n.d. 0.018±0.002b n.d. n.d. 0.056±0.001b 0.071±0.0
n.d. 0.047±0.003bc n.d. n.d. n.d. n.d. 0.026±0.001ab n.d. n.d. 0.021±0.002c 0.047±0.0
h n.d. 0.044±0.002c n.d. n.d. n.d. n.d. 0.022±0.005b n.d. n.d. 0.021±0.003c 0.044±0.0
0.007±0.001a 0.606±0.030a 0.030±0.002a 0.030±0.001a 0.012±0.000a 0.006±0.000a 0.039±0.004a n.d. n.d. 0.067±0.002a 0.685±0.0
0.006±0.001a 0.315±0.021b 0.013±0.002b 0.026±0.001b 0.013±0.002a 0.006±0.003a 0.020±0.001b n.d. n.d. 0.055±0.004b 0.372±0.0
0.004±0.000b 0.216±0.007c 0.016±0.001b 0.012±0.002c 0.011±0.002a n.d. 0.028±0.006b n.d. n.d. 0.021±0.001d 0.256±0.0
h 0.004±0.000b 0.216±0.006c 0.013±0.000b 0.014±0.002c 0.013±0.001a 0.006±0.001a 0.025±0.002b n.d. n.d. 0.039±0.001c 0.262±0.0
0.010±0.002a 0.781±0.020a 0.035±0.001a 0.033±0.001a 0.015±0.002a 0.006±0.001a 0.110±0.006a 0.019±0.001a 0.084±0.006a 0.438±0.004a 0.870±0.0
0.004±0.000b 0.124±0.008b n.d. 0.011±0.002b 0.007±0.001b n.d. 0.036±0.004c n.d. 0.015±0.001b 0.115±0.011b 0.141±0.0
0.004±0.001b 0.144±0.001b 0.007±0.002b 0.009±0.002b 0.009±0.002b n.d. 0.087±0.002b n.d. 0.015±0.002b 0.071±0.003c 0.169±0.0
h n.d. 0.126±0.006b 0.006±0.001b 0.008±0.003b 0.007±0.002b n.d. 0.086±0.006b n.d. n.d. 0.074±0.013c 0.147±0.0
n.d. 0.332±0.006a 0.017±0.001a 0.020±0.001a 0.008±0.002a n.d. 0.063±0.001a n.d. n.d. 0.170±0.008a 0.377±0.0
0.005±0.001a 0.178±0.010b n.d. 0.023±0.002a 0.010±0.002a n.d. 0.033±0.001b n.d. n.d. 0.146±0.013b 0.211±0.0
0.004±0.000a 0.119±0.002c n.d. 0.009±0.002b 0.007±0.001a n.d. 0.043±0.006b n.d. n.d. 0.066±0.002c 0.135±0.0
h n.d. 0.119±0.002c n.d. 0.010±0.000b 0.007±0.001a n.d. 0.044±0.012b n.d. n.d. 0.071±0.002c 0.135±0.0
0.010±0.012a 0.615±0.454a 0.030±0.027a 0.031±0.019a 0.014±0.010a 0.005±0.005a 0.061±0.025a 0.010±0.016a 0.033±0.050a 0.244±0.258a 0.695±0.5
0.005±0.004b 0.205±0.114b 0.008±0.008b 0.020±0.011b 0.011±0.007b 0.005±0.005a 0.033±0.013c n.d. 0.011±0.016b 0.107±0.078b 0.249±0.1
0.002±0.004bc 0.145±0.076b 0.009±0.008b 0.011±0.007c 0.009±0.005bc 0.002±0.004b 0.054±0.029ab n.d. 0.003±0.007b 0.053±0.034b 0.175±0.0
h 0.001±0.003c 0.132±0.071b 0.007±0.006b 0.010±0.006c 0.007±0.005c 0.003±0.004b 0.047±0.026b n.d. 0.004±0.007b 0.050±0.028b 0.160±0.0
53.6 33.3 26.4 63.5 80.5 98.3 54.0 n.d. 34.2 43.9 35.8
22.8 23.6 28.4 34.4 62.4 47.7 89.0 n.d. 10.4 21.7 25.2
h 13.9 21.5 23.1 34.0 51.7 66.3 77.6 n.d. 11.2 20.6 23.0
 29

46.4 66.7 73.6 36.5 19.5 1.7 46.0 n.d. 65.8 56.1 64.2
77.2 76.4 71.6 65.6 37.6 52.3 11.0 n.d. 89.6 78.3 74.8
h 86.1 78.5 76.9 66.0 48.3 33.7 22.4 n.d. 88.8 79.4 77.0
646 n.d.  not detectable; STD  standard deviations; values in columns marked with different

647 letters are statistically different at p  0.05; *average of all analyzed varieties and genotypes

648

649 Highlights

650  Carotenoid changes were assessed in color-grained wheat flours during bun-making
651  Stability of carotenoids was not related to grain color, but was dependent on genotype
652  Most destructive was the dough preparation; the heat treatment had a lower effect
653  All-E-lutein decreased more than Z-forms due to its isomerization
654  Esterified carotenoids degraded to a similar extent as the free carotenoids
655

656