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Accepted Manuscript

Static Headspace Analysis of Odor ants in Commer cial Rice Pr oteins

Jing Zhao, William L. Boatright

PII: S0308-8146(16)31739-3
DOI: http://dx.doi.org/10.1016/j.foodchem.2016.10.086
Reference: FOCH 20074

To appear in: Food Chemistry

Received Date: 16 May 2016


Revised Date: 13 October 2016
Accepted Date: 19 October 2016

Please cite this article as: Zhao, J., Boatright, W.L., Static Headspace Analysis of Odor ants in Commer cial Rice
Pr oteins, Food Chemistry (2016), doi: http://dx.doi.org/10.1016/j.foodchem.2016.10.086

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1 Static Headspace Analysis of Odorants in Commercial Rice Proteins

3 Jing Zhao1,2, William L. Boatright1,


1
4 Department of Animal and Food Sciences, University of Kentucky, Lexington, KY, 40546,

5 USA
2
6 Food Science and Technology Program, School of Kinesiology and Nutritional Science,

7 California State University, Los Angeles, Los Angeles, CA, 90032, USA

9 * Corresponding author:

10 Prof. William L. Boatright

11 University of Kentucky

12 412 W.P. Garrigus Building

13 Lexington, KY 40546-0215

14 Phone: 859-257-5988

15 Email: wlboat1@email.uky.edu

16

17 Dr. Jing Zhao

18 Email: jzhao30@calstatela.edu

19 Dr. Zhao has moved to California State University, Los Angeles after the research was

20 completed at the University of Kentucky.

21

22 Manuscript prepared for Food Chemistry


23 Abstract

24 Accurate identification of the odor-contributing compounds in aqueous slurries of rice

25 proteins is necessary to improve their overall flavor characteristics. The objective of this study

26 was to identify the primary odorants in rice protein slurries using static headspace analysis. Five

27 commercial rice protein (RP) products, RP-G, RP-O, RP-RM, RP-RS1, and RP-RS2, were

28 analyzed. RP-G contained the lowest levels of most of the odorants. Acetaldehyde was present in

29 the highest amount in RP-O (0.434 mg/m3). RP-RM had the highest levels of hexanal (5.907

30 mg/m3), methanethiol (0.138 mg/m3), pentanal (1.575 mg/m3), and 2-pentylfuran (5.702 mg/m3).

31 Corresponding odor values were, 111, 86, 22 and 21, respectively. In RP-RS1 and RP-RS2, the

32 predominant odorants were dimethyl disulfide, dimethyl trisulfide, and hexanal. The results

33 showed the importance of the volatile compounds produced from amino acids, including the

34 sulfur-containing compounds and acetaldehyde, as well as lipid oxidation derived odorants to the

35 overall odor of rice proteins.

36

37 Keywords

38 Gas chromatography-mass spectrometry; sulfur-containing odorants; acetaldehyde; manganese;


39 copper; iron; sulfite; hydroperoxides.

40

41 Chemical compounds studied in this article:

42 Acetaldehyde (PubChem CID: 177); Benzaldehyde (PubChem CID: 240); Dimethyl disulfide

43 (PubChem CID: 12232); Dimethyl trisulfide (PubChem CID: 19310); Hexanal (PubChem CID:

44 6184); Hydrogen sulfide (PubChem CID: 402); Methanethiol (PubChem CID: 878); Pentanal

45 (PubChem CID: 8063); and 2-Pentylfuran (PubChem CID: 19602).

46 Highlights
47 The primary rice protein odorants were identified using static headspace analysis.

48 Three sulfur-containing compounds were identified among the primary odorants.

49 Acetaldehyde contributed significantly to the odor of RP-O but not other samples.

50 The major lipid-derived odorants included hexanal, pentanal, and 2-pentayl furan.

51 Metals, sulfite, and hydroperoxides were detected in rice protein samples.

52

53
54 1. Introduction

55 Rice is composed of 79% of protein (USDA 2015). It is a hypo-allergic (Reche et al.,

56 2010) and gluten-free protein product that could promote weight loss and muscles building. Due

57 to its hypo-allergic property, rice protein has been used to replace soy protein in infant formula

58 (Zhao et al., 2014). Because of the irrigation practices used in growing rice, improved water

59 management practices are necessary to increase productivity and conserve important natural

60 resources (Bouman and Tuong, 2001). This is particularly true in Africa (Viala, 2008). Rice

61 protein has also been used in health drinks, cereal snack foods, baked foods, meat products, and

62 healthcare products. However, it has an odor characteristic that needs to be improved to meet

63 consumer acceptability. Accurate identification of the odor-contributing compounds is necessary

64 before the undesirable odorants can be minimized.

65 Many chemicals, naturally present in rice proteins or added to rice proteins during

66 processing, could affect the odorants formation. Sulfite has been used in the solubilization and

67 extraction of rice bran protein due to its capacity to increase the pH and facilitate the cleavage of

68 disulfide linkages (Hamada, 1999). Sulfite has been demonstrated to be important in the

69 formation of the odor compound methanethiol in soy protein (Boatright, Lei, & Stine, 2006; Lei

70 & Boatright, 2007). A previous study showed that addition of 3.2 mM sodium sulfite resulted in

71 a 5-fold increase in methanethiol in aqueous slurries of soy protein isolate even though sulfite

72 does not directly contribute sulfur for methanethiol (Lei & Boatright, 2006). Naturally occurring

73 lipoxygenases are also thought to play an important role in the formation of flavor and aroma in

74 many plant products (Suzuki & Matsukura, 1997). Williams, Lim, Chen, Pangborn, and

75 Whitaker (1986) reported that lipoxygenase was the primary cause of development of off-flavors

76 in English green peas and green beans. Another study reported that a number of volatile
77 compounds, including pentanol, hexanol, heptanol, hexanal, 3-cis-hexenal, 2-propanone, 2-

78 pentylfuran, and ethyl vinyl ketone, could be formed via lipoxygenase-catalyzed oxidation of the

79 unsaturated lipid present in soy and remain in soy protein concentrate and isolate due to their

80 association with proteins (O'Keefe, Wilson, Resurreccion, & Murphy, 1991). Besides, lipid

81 oxidation products have also been shown to form in soy protein isolates after the powder is

82 hydrated (Boatright & Lu, 2007). Minerals also play a role in odorants formation in proteins

83 powders. It was reported that in the presence of manganese, sulfite, and oxygen, methanethiol

84 could be formed spontaneously in soy proteins from methionine. Also, manganese was the most

85 active metal in catalyzing methanethiol formation from methionine and sulfite, followed by

86 copper and iron (Lei et al., 2007). Methionine was shown to be the methyl group donor for

87 sulfite-associated methanethiol formation (Lei et al., 2006). The methionine content in rice was

88 reported to vary from 1.5% to 3.5% with an average of about 2.1% (Lasztity, 1995) which is

89 significantly higher than that in soy (0.92 %).

90 The odor active compounds in soy proteins (Aaslyng, Elmore, & Mottram, 1998; Ang &

91 Boatright, 2003), pea proteins (Murat, Bard, Dhalleine, & Cayot, 2013), and hydrolyzed rice

92 proteins (Jarunrattanasri, Theerakulkait, & Cadwallader, 2007; Kaewka, Therakulkait, &

93 Cadwallader, 2011) have been investigated. Previous study on rice protein odorants used solvent

94 assisted flavor evaporation (SAFE) technique which causes a loss of low molecular weight

95 compounds and the concentration of the higher molecular weight compounds during distillation.

96 A representative overall composition of the primary odorants in rice proteins has not been

97 documented using static headspace analysis. The current investigation differs from previous

98 investigations by providing the concentrations of key odorants in the sample headspace (mg/m3

99 of air) and their calculated odor values in air. The objective of this study was to characterize the

100 types and amounts of primary odorants that contribute to the flavor of rice proteins using static
101 headspace analysis. The presence of metals, sulfite, and hydroperoxides was also documented

102 and their influence on odorants formation was discussed.

103

104 2. Materials and methods

105

106 2.1 Materials

107 Five rice protein products from commercial sources were investigated in this study. They

108 include 1) RP-RM, a non-soluble rice protein concentrate, 2) RP-O, a brown rice protein

109 extracted without hexane, and three sprouted brown rice proteins 3) RP-G, 4) RP RS 1 and 5)

110 RP RS2. Standard chemicals including hexanal, dimethyl trisulfide, methanethiol, and

111 acetaldehyde were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Chemical 2-

112 pentylfuran was purchased from Bedoukian Research, Inc. (Danbury, CT). Other analytical

113 grade chemicals were purchased from Sigma Chemical Co. and Fisher Scientific (Indianapolis,

114 IN, USA).

115

116 2.2 Proximate analysis of rice proteins

117 Moisture content was determined by measuring the weight loss after drying a sample of

118 about 5 g in a 105 C oven for 1618 h according to AOAC official method 2008.06 (AOAC,

119 2004). Total crude fat was determined using SER 148 solvent extractors (VELP Scientifica Srl,

120 Usmate Velate MB, Italy) with petroleum ether as the solvent. The plate heating temperature was

121 set to 110 C. Samples were socked and flushed for one hour, respectively. After the solvent was

122 evaporated, the cups were dried at 100 C for 30 min. Ash content was determined by

123 incinerating about 1 g of the protein samples in a muffle furnace at 580 C for 48 h. Protein
124 content was analyzed using a vario MAX CN micro elemental analyzer (Elementar

125 Analysensystem GmbH, Hanau, Germany). According to Food and Agricultural Organization of

126 the United Nations (FAO, 2003), a conversion factor of 5.95 was used to calculate rice protein

127 content from nitrogen. Protein analysis was performed with two replications. All other

128 measurements were repeated three times.

129

130 2.3 Elemental analysis of rice proteins

131 Manganese, copper, and iron contents in rice proteins were analyzed using an AAnalyst

132 200 atomic absorption spectrometer (PerkinElmer Inc., Waltham, MA, USA). Samples were dry

133 ashed followed by wet decomposition. About 1 g of protein sample was weighed into a crucible

134 and ashed in an ash oven at 580 C for 48 h. The residue was solubilized in 5 mL 6 N HCl,

135 heated on a hot plate to evaporate, and then re-solubilized and soaked for one hour in 10 mL 0.1

136 N HNO3. The resulting solution was quantitatively transferred and diluted to 25 mL with

137 deionized water in a volumetric flask. Manganese, copper, and iron were analyzed at 279.48 nm

138 (slit 1.8/0.6 mm), 324.75 nm (slit 2.7/0.8 mm), and 248.33 nm (slit 1.8/1.35 mm), respectively.

139 The mineral concentrations in the rice proteins were calculated based on a standard curve

140 analyzed along with the samples.

141

142 2.4 Sulfite analysis

143 The amount of sulfite in rice proteins was analyzed using the optimized Monier-Williams

144 method according to AOAC official method 990.28 (AOAC, 2004) with modifications. A 400-

145 mm Davies-type condenser (Ace Glass Co., Louisville, KY, USA) was used instead of the 300-
146 mm Allihn condenser. A bell-shaped defoamer was connected between the 1000 mL flask and

147 the condenser to block foams.

148

149 2.5 Protein hydroperoxides determination

150 Protein hydroperoxides were measured using ferric-xylenol orange (FOX) 1 assay

151 according to Wolff (1994). FOX reagent was prepared as 100 M xylenol orange, 250 M

152 ammonium ferrous sulfate, and 100 mM sorbitol in 25 mM H2SO4. Iron salt needs to be

153 dissolved in the acid directly. One gram of rice protein sample was solubilized in 19 mL d.d.

154 H2O (5%) and stirred at room temperature for 30 min. Sample solution of 100 L was mixed

155 with 1900 L of FOX reagent, vortexed and incubated at room temperature for 30 min. Then

156 after centrifugation at 2000 rpm for 5 min, the absorbance was recorded at 560 nm. A standard

157 curve was prepared using 010 M hydrogen peroxide. The sample hydroperoxide

158 concentrations were calculated based on the standard curve and expressed as mol H2O2

159 equivalents/g protein.

160

161 2.6 Gas chromatography-olfactometry-mass spectrometry (GC-O-MS)

162 Rice protein slurries (5%, w/v) were prepared in a 1 L flask sealed with a septum. The

163 protein slurry was stirred for 40 min at room temperature before 15 mL of the headspace was

164 withdrawn using a 25 mL gas-tight syringe (preheated to 45 C) and analyzed using GC-O-MS.

165 The GC-MS system is composed of an Agilent model 6890N gas chromatograph, a 5973 mass

166 spectrometer, a ms-NoVentTM system (SGE International, Ringwood, Australia), and an indirect

167 liquid-nitrogen trap (SGE International) at the beginning of the column to cryofocus volatile

168 analytes. The temperature of the trap was monitored using a type J thermocouple thermometer
169 (Glas-Col, Terre Haute, IN). Before sample injection, the liquid-nitrogen trap was cooled to

170 below 85 C to ensure the trap of low boiling point compounds such as hydrogen sulfite,

171 methanethiol, and acetaldehyde which have a boiling point of 60 C, 5.95 C and 20.2 C,

172 respectively. The ms-NoVentTM was turned on. The valve for septum purge and split vent was

173 closed. Fifteen milliliters of the sample headspace was injected at a rate of 5 mL/min. The valve

174 for septum purge and split vent was opened after 2 min and then in another 2 min, the ms-

175 NoVentTM was turned off. The nitrogen flow to the cryotrap was turned off right before the GC

176 run was started. The column was a HP-5MS capillary column with the dimensions of 30 m 530

177 m 1.5 m (Agilent Technologies, Palo Alto, CA, USA). The flow rate of helium (carrier gas)

178 through the columns was 2.5 mL/min, with 1.5 mL/min emerging from the sniff port (SGE

179 International) and 1 mL to the mass spectrometer. The column temperature was held at 35 C for

180 4 min, increased at 10 C/min to 250 C and held there for 5 min. The injection port temperature

181 was maintained at 165 C.

182 Volatile compounds were identified by comparison of mass spectra to a spectral database

183 (NIST05) (ChemSW, Inc., Fairfield, CA, USA), comparison of retention times to that of

184 authentic standards, and comparison of olfactory response to authentic standards. The amount of

185 the primary odor compounds were quantified as previously described (Boatright, 2002) using

186 ethanethiol as an internal standard. The retention time, standard curves, and published odor

187 threshold values for the primary odorants are shown in Table 1. Odor values of the primary

188 odorants were calculated as:

odorant concentration in mg/m


Odor value =
odor threshold in mg/m

189

190 2.7 Statistical analysis


191 All the analyses were repeated three times unless specified. Statistical analysis was

192 performed using Statistix software 9.0 (Analytical Software, Tallahassee, FL, USA). A linear

193 model was used for the overall analysis of variance. When a significant treatment effect was

194 found (P < 0.05), multiple comparison was performed using Tukey HSD all-pairwise comparison

195 test (P < 0.05).

196

197 3. Results and Discussion

198

199 3.1 Proximate analysis of rice proteins

200 The proximate compositions of the commercial rice proteins are presented in Table 2.

201 The protein powders had a moisture content of 2.14.6%, a protein content of 7484%, a fat

202 content of 0.045.3%, and an ash content of 0.92.4%. The sprouted rice proteins (RP-RS1, RP-

203 RS2, and RP-G) had lower moisture contents and higher protein and fat contents than RP-RM

204 and RP-O (P < 0.05). Among these rice proteins, RP-G was especially high in fat (5.27%) and

205 RP-O was substantially low in fat (0.04%) but high in ash (2.31%).

206

207 3.2 Elemental analysis of rice proteins

208 The manganese, copper, and iron contents in rice protein samples are shown in Figure 1.

209 The brown rice protein RP-O had the highest levels of manganese and iron, which contributes to

210 its high ash content. As reported, the mineral contents are high in brown rice which contains the

211 mineral-rich outer bran layer of the rice grain (Patil & Khan, 2011). Two sprouted brown rice

212 proteins, RP-RS1 and RP-RS2, had the highest copper content and the second highest manganese

213 and iron contents among the rice protein samples.


214 As shown in Figure 1, the manganese content varied from 1.6 ppm in RP-RM to 58.8

215 ppm in RP-O. RP-O had a manganese content ten times higher than the sample with the next

216 highest manganese level (58.8 ppm versus 5.5 ppm in RP-RS2), which is significant higher than

217 all the other rice protein samples (P < 0.5). The manganese content in rice was reported to range

218 from 15.7 to 23.3 ppm in different rice varieties and was about 17 ppm in brown rice (Anjum,

219 Pasha, Bugti, & Butt, 2007). The manganese content in wild rice and cultivated brown rice was

220 reported to be 14 ppm and 30-39 ppm, respectively. Another study showed that the manganese

221 content in full rice flour could be as high as 47 ppm (Shih & Daigle, 2000). In the process of

222 protein extraction, the manganese content could be reduced to about 1.4 ppm. The reported

223 manganese content in rice protein was very close to the manganese content detected in RP-RM.

224 Based on the reported manganese content in rice, RP-O was the only rice protein where

225 manganese was concentrated during protein extraction.

226 There was a much smaller variation in copper content among these rice protein samples,

227 from 18.9 ppm to 27.2 ppm. The reported copper content in rice ranges from 5.8 to 9.2 ppm

228 (Anjum et al., 2007). The current results showed a higher copper content in rice proteins,

229 suggesting a copper-concentrating effect of protein extraction process.

230 According to USDA National Nutrient Database for Standard Reference (USDA 2015),

231 the iron content is 12.919.6 ppm in raw brown and wild rice and 8 ppm in raw, unenhanced

232 white rice. The current results showed that the iron contents in rice proteins varied from 25.0

233 ppm in RP-RM to 208.4 ppm in RP-O. Interestingly, the iron contents in rice protein samples

234 showed a positive correlation with their manganese contents (R = 0.82, linear regression).

235

236 3.3 Sulfite analysis


237 Sulfite has been used in the solubilization and extraction of rice bran protein due to its

238 capacity to increase the pH and facilitate the cleavage of disulfide linkages (Hamada, 1999).

239 However, to the best of our knowledge, the residual sulfite content in rice protein products has

240 not been documented. In this study, the total sulfite content in commercial rice protein products

241 was measured using the AOAC optimized Monier-Williams method and the results are shown in

242 Figure 2. The current results showed that the rice protein products contained 3.15.6 ppm sulfite.

243 The intrinsic sulfite content in commercial soy protein isolate analyzed using the same method

244 was reported to be 2231 ppm (Stine, Boatright, & Lu, 2004), which is about six to seven times

245 higher than the total sulfite content in rice detected in this study. In spite of the small amount,

246 reaction of residual sulfite (SO32) with oxidizing reagents, such as hydroperoxides (Erben-Russ,

247 Bors, Winter, & Saran, 1986), or transition metals and oxygen (Ermakov, 2002; Lei et al., 2007),

248 could produce sulfite radical anion (SO3), a chain carrier in the aerobic free-radical chain

249 oxidation of sulfur dioxide in aqueous solution. Further reaction of the sulfite radicals with

250 transition metals and oxygen could generate sulfate radical anions which are capable of initiating

251 the formation of methionine radical cation (Choi et al., 2006) and thus the formation of the odor

252 compound methanethiol (Boatright et al., 2006). Sulfite and sulfate free-radicals that contribute

253 to the degradation of the essential amino acids in proteins not only alter the functional and

254 nutritional characteristics of the protein but also influence the protein flavor characteristics.

255

256 3.4 Protein hydroperoxides determination

257 Protein hydroperoxides measured using the FOX 1 assay is shown in Figure 3. FOX

258 assay is based on the ability of hydroperoxides to react with an excess of Fe2+ at low pH in the

259 presence of xylenol orange (XO) dye. The amount of Fe3+ generated by this reaction is measured
260 as a Fe-XO complex at 560 nm (Gay, Collins, & Gebicki, 1999). FOX 1 assay measures low

261 amount of water soluble hydroperoxides with high sensitivity (Wolff, 1994). Since the rice

262 protein samples all contained low fat content (< 5.3 %), the FOX 2 assay, which is suitable for

263 lipid soluble hydroperoxides, gave low readings and was not suitable for the samples in this

264 study (results not shown). The rice protein samples contained 0.04 0.15 mol

265 hydroperoxides/gram protein sample (Figure 3). The hydroperoxide contents were high in the

266 three sprouted brown rice proteins. RP-RS1 and RP-RS2 contained the highest levels of

267 hydroperoxides followed by RP-G. RP-O and RP-RM contained significantly lower levels of

268 hydroperoxides than the sprouted brown rice proteins (P < 0.05), indicating an influence of the

269 sprouting process on hydroperoxide formation and stabilization in extracted rice proteins. The

270 significance of hydroperoxides and their degradation products on the taste and odor of proteins

271 have been recognized for decades (Hamdy, 1974). Generally, reactive hydroperoxide groups

272 could be formed in high yields on proteins exposed to a wide range of reactive oxygen species

273 (Gebicki, Du, Collins, & Tweeddale, 2000). Hydroperoxides could degrade to form volatile

274 compounds such as aldehydes, ketones, and alcohols and thus influence the flavor of the protein

275 products during storage.

276

277 3.5 Gas chromatography-olfactometry-mass spectrometry (GC-O-MS)

278 The primary odorants in rice protein samples were analyzed using static headspace

279 analysis in this study. Comparing to solvent assisted flavor evaporation (SAFE) technique, which

280 causes a loss of low molecular weight compounds and the concentration of the higher molecular

281 weight compounds during distillation, static headspace analysis enables the characterization of a

282 representative overall composition of the primary odorants. However, sensitivity is limited in
283 static headspace analysis and thus it can only detect and quantify the most abundant and intense

284 odorants (Alasalvar, Taylor, & Shahidi, 2005).

285 The amounts and odor values of the primary odorants in rice protein samples are shown

286 in Table 3. The primary volatile compounds contributing to the odor of rice proteins varied

287 substantially from one product to another. The volatile compounds that were present in an

288 amount above their reported odor threshold values included acetaldehyde, sulfur-containing

289 compounds (methanethiol, dimethyl disulfide, and dimethyl trisulfide), and lipid oxidation

290 derived odorants (hexanal, pentanal, 2-pentylfuran, and benzaldehyde). Hexanal concentration in

291 the headspace of 5% rice protein slurries varied from 0.54 mg/m3 in RP-O to 5.91 mg/m3 in RP-

292 RM, where the odor value of hexanal was 111. Besides the high hexanal concentration, RP-RM

293 also had the highest levels of methanethiol (0.138 mg/m3, OV=86), pentanal (1.575 mg/m3,

294 OV=22), and 2-pentylfuran (5.702 mg/m3, OV=21) among the five tested rice protein samples.

295 Even though RP-RM has a very low solubility (data not shown), it did not influence the release

296 of the odor compounds from the protein slurry. In RP-RS1 and RP-RS2, the predominant

297 odorants included dimethyl disulfide (1.522 and 1.655 mg/m3, OV=52 and 57, respectively),

298 dimethyl trisulfide (0.042 and 0.048 mg/m3, OV=35 and 40, respectively), hexanal (2.171 and

299 2.201 mg/m3, OV=50 and 51, respectively), and pentanal (0.480 and 0.509 mg/m3, OV=14 and

300 15, respectively). Among these, the two RP-RS rice proteins were especially high in dimethyl

301 disulfide and dimethyl trisulfide when compared to other samples. Acetaldehyde was present in

302 RP-O at the level of 0.434 mg/m3 (OV=5), which was significantly higher than its concentration

303 in other rice protein products. It has been reported that acetaldehyde could be derived from

304 thermal degradation of amino acids such as serine, cysteine, aspartic acid, alanine, and threonine

305 (Perez Locas & Yaylayan, 2004). For example, acetaldehyde could be formed from serine and
306 cysteine through decarboxylation (formation of ethanolamine) followed by loss of a molecule of

307 ammonia. It could also be generated from alanine through Strecker reaction; and in turn alanine

308 could be generated from aspartic acid through decarboxylation (Perez Locas et al., 2004).

309 Besides acetaldehyde, dimethyl trisulfide (0.021 mg/m3, OV=18), dimethyl disulfide (0.340

310 mg/m3, OV=12), hexanal (0.540 mg/m3, OV=10), and methanethiol (0.014 mg/m3, OV=8) also

311 contributed markedly to the odor of RP-O. RP-G contained the lowest levels of most of the

312 odorants. The most intense odorants in RP-G were hexanal (1.802 mg/m3, OV=34), dimethyl

313 disulfide (0.530 mg/m3, OV=18), and dimethyl trisulfide (0.018 mg/m3, OV=15). In general, the

314 three sprouted brown rice proteins, RP-G, RP-RS1, and RP-RS2, contained the highest levels of

315 dimethyl disulfide. It is worth noting that RP-G, which had the lowest total sulfite level (Figure

316 2), contained the lowest levels of methanethiol and dimethyl trisulfide of all the products (Table

317 3).

318 The results from this study confirmed the importance of the sulfur-containing compounds

319 as well as the lipid oxidation products to the overall odor of rice proteins. In this study, an

320 indirect liquid-nitrogen trap was configured at the beginning of the column to cryofocus volatile

321 odorants. This is the first time low boiling point volatile compounds such as hydrogen sulfide

322 and methanethiol have been considered, even though hydrogen sulfide was not detected in any of

323 the rice protein products. This is also the first time that methanethiol, dimethyl disulfide, and

324 acetaldehyde are reported in rice protein products. In order to prevent or otherwise eliminate the

325 formation of primary odor compounds in rice protein products, it is critical to first know what

326 compounds are the major contributors. Based on the current findings, future studies could be

327 performed to address the chemical mechanisms responsible for their formation and the possible

328 means to minimize their occurrences.


329

330 4. Conclusions

331 This is the first time that metals, sulfites, hydroperoxides and volatile odorants such as

332 methanethiol, dimethyl disulfide, and acetaldehyde have been considered and reported in rice

333 protein products. No direct correlation has been found between the amount of odorants formed

334 and the content of metal, sulfites, and hydroperoxides. However, the presence of these substrates,

335 catalysts, and potential radicals suggests potential mechanisms of the odorants formation. Future

336 study is needed to substantiate these potential odorants formation paths.

337

338 Acknowledgements

339 The information reported in this paper (No. 16-07-054) is part of a project of the

340 Kentucky Agricultural Experiment Station and is published with the approval of the Director.

341

342 Conflict of Interest

343 The authors certify that they have NO affiliations with or involvement in any

344 organization or entity with any financial interest (such as honoraria; educational grants;

345 participation in speakers bureaus; membership, employment, consultancies, stock ownership, or

346 other equity interest; and expert testimony or patent-licensing arrangements), or non-financial

347 interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the

348 subject matter or materials discussed in this manuscript.

349

350 References
351 Aaslyng, M. D., Elmore, J. S., & Mottram, D. S. (1998). Comparison of the aroma characteristics

352 of acid-hydrolyzed and enzyme-hydrolyzed vegetable proteins produced from soy.

353 Journal of Agricultural and Food Chemistry, 46(12), 5225-5231.

354 Alasalvar, C., Taylor, K. A., & Shahidi, F. (2005). Comparison of volatiles of cultured and wild

355 sea bream (Sparus aurata) during storage in ice by dynamic headspace analysis/gas

356 chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry, 53(7),

357 2616-2622.

358 Ang, E. C., & Boatright, W. L. (2003). Olfactory perception of major odorants found in the

359 headspace of aqueous soy protein isolate slurries. Journal of Food Science, 68(1), 388-

360 393.

361 Anjum, F., Pasha, I., Bugti, M. A., & Butt, M. (2007). Mineral composition of different rice

362 varieties and their milling fractions. Pakistan Journal of Agricultural Sciences, 44(2),

363 332-336.

364 AOAC (Association of Official Analytical Chemists) International (2004). Official Methods of

365 Analysis, 16th Edition. AOAC International, Gaithersburg, MD.

366 Boatright, W. L. (2002). Effect of gallic acid on the aroma constituents of soymilk and soy

367 protein isolates. Journal of the American Oil Chemists Society, 79(4), 317-323.

368 Boatright, W. L., Lei, Q. X., & Stine, C. J. (2006). Sulfite formation in isolated soy proteins.

369 Journal of Food Science, 71(3), C115-C119.

370 Boatright, W. L., & Lu, G. (2007). Hexanal synthesis in isolated soy proteins. Journal of the

371 American Oil Chemists Society, 84(3), 249-257.

372 Bouman, B.A.M., & Tuong, T.P., (2001). Field water management to save water and increase its

373 productivity in irrigated lowland rice. Agricultural Water Management, 49:11-30.


374 Choi, J., Sullards, M. C., Olzmann, J. A., Rees, H. D., Weintraub, S. T., Bostwick, D. E.,

375 Gearing, M., Levey, A. I., Chin, L.-S., & Li, L. (2006). Oxidative damage of DJ-1 is

376 linked to sporadic Parkinson and Alzheimer diseases. Journal of Biological Chemistry,

377 281(16), 10816-10824.

378 Erben-Russ, M., Bors, W., Winter, R., & Saran, M. (1986). The reaction of sulfite anion radical

379 (SO.-3) with polyunsaturated fatty acids. International Journal of Radiation Applications

380 and Instrumentation. Part C. Radiation Physics and Chemistry, 27(6), 419-424.

381 Ermakov, A. N., & Purmal, A. P. (2002). Catalysis of HSO3/SO32 oxidation by manganese ions.

382 Kinetics and Catalysis, 43(2), 249-260.

383 Gay, C., Collins, J., & Gebicki, J. M. (1999). Hydroperoxide assay with the ferricxylenol

384 orange complex. Analytical Biochemistry, 273(2), 149-155.

385 Gebicki, J. M., Du, J., Collins, J., & Tweeddale, H. (2000). Peroxidation of proteins and lipids in

386 suspensions of liposomes, in blood serum, and in mouse myeloma cells. Acta Biochimica

387 Polonica, 47, 901-911.

388 Hamada, J. S. (1999). Use of proteases to enhance solubilization of rice bran proteins 1. Journal

389 of Food Biochemistry, 23(3), 307-321.

390 Hamdy, M. (1974). Nutritional aspects in textured soy proteins. Journal of the American Oil

391 Chemists Society, 51(1), 85A-90A.

392 Jarunrattanasri, A., Theerakulkait, C., & Cadwallader, K. R. (2007). Aroma components of acid-

393 hydrolyzed vegetable protein made by partial hydrolysis of rice bran protein. Journal of

394 Agricultural and Food Chemistry, 55(8), 3044-3050.


395 Kaewka, K., Therakulkait, C., & Cadwallader, K. R. (2011). Effect of drying and proline

396 addition on aroma volatile compounds of acid-hydrolysed rice bran protein concentrate.

397 International Journal of Food Science and Technology, 46(8), 1654-1660.

398 Lasztity, R. (1995). The chemistry of cereal proteins: CRC press.

399 Lei, Q., & Boatright, W. L. (2006). Methionine is the methyl group donor for sulfite-associated

400 methanethiol formation in isolated soy proteins. Journal of Food Science, 71(9), C527-

401 C531.

402 Lei, Q., & Boatright, W. L. (2007). Sulfite-radical anions in isolated soy proteins. Journal of

403 Food Science, 72(5), C300-C306.

404 Murat, C., Bard, M.-H., Dhalleine, C., & Cayot, N. (2013). Characterisation of odour active

405 compounds along extraction process from pea flour to pea protein extract. Food Research

406 International, 53(1), 31-41.

407 O'Keefe, S. F., Wilson, L. A., Resurreccion, A. P., & Murphy, P. A. (1991). Determination of the

408 binding of hexanal to soy glycinin and. beta.-conglycinin in an aqueous model system

409 using a headspace technique. Journal of Agricultural and Food Chemistry, 39(6), 1022-

410 1028.

411 Patil, S. B., & Khan, M. K. (2011). Germinated brown rice as a value added rice product: A

412 review. Journal of Food Science and Technology, 48(6), 661-667.

413 Perez Locas, C., & Yaylayan, V. A. (2004). Origin and mechanistic pathways of formation of the

414 parent furan A food toxicant. Journal of Agricultural and Food Chemistry, 52(22), 6830-

415 6836.

416 Pohanish, R. P. (2012). Sittigs handbook of toxic and hazardous chemicals and carcinogens.

417 Sixth edition. Elsevier Publications, Oxford, UK.


418 Reche, M., Pascual, C., Fiandor, A., Polanco, I., RiveroUrgell, M., Chifre, R., Johnston, S., &

419 MartnEsteban, M. (2010). The effect of a partially hydrolysed formula based on rice

420 protein in the treatment of infants with cows milk protein allergy. Pediatric Allergy and

421 Immunology, 21(4p1), 577-585.

422 Ruth, J. H. (1986). Odor thresholds and irritation levels of several chemical substances: a review.

423 American Industrial Hygiene Association Journal, 47(3), A-142-A-151.

424 Rychlik, M., Schieberle, P., & Grosch, W. (1998). Compilation of odor thresholds, odor

425 qualities and retention indices of key food odorants: Dt. Forschungsanst. fr

426 Lebensmittelchemie.

427 Shih, F. F., & Daigle, K. W. (2000). Preparation and characterization of rice protein isolates.

428 Journal of the American Oil Chemists Society, 77(8), 885-889.

429 Stine, C. J., Boatright, W. L., & Lu, G. (2004). Intrinsic sulfite content of isolated soy proteins.

430 Journal of the American Oil Chemists Society, 81(9), 829-833.

431 Suzuki, Y., & Matsukura, U. (1997). Lipoxygenase activity in maturing and germinating rice

432 seeds with and without lipoxygenase-3 in mature seeds. Plant Science, 125(2), 119-126.

433 Van Gemert, L. (2003). Odour thresholds-compilations of odour thresholds in air, water and

434 other media. Oliemans Punter & Partners BV, The Netherlands.

435 Verschueren, K. (1983). Handbook of environmental data on organic chemicals. Second edition.

436 Van Nostrand Reinhold Co. Inc., New York, NY

437 Williams, D. C., Lim, M. H., Chen, A. O., Pangborn, R. M. and Whitaker, J. R. (1986).

438 Blanching of vegetables for freezing: which indicator enzyme to choose. Food

439 Technology, 40, 130-140.


440 Wolff, S. P. (1994). [18] Ferrous ion oxidation in presence of ferric ion indicator xylenol orange

441 for measurement of hydroperoxides. Methods in Enzymology, 233, 182-189.

442 Zhao, Q., Selomulya, C., Xiong, H., Chen, X. D., Li, X., Wang, S., Bai, C., Peng, H., Zhou, Q.,

443 & Sun, W. (2014). Rice dreg protein as an alternative to soy protein isolate: comparison

444 of nutritional properties. International Journal of Food Properties, 17(8), 1791-1804.

445 EPA (United States Environmental Protection Agency). (2000).

446 https://www3.epa.gov/airtoxics/hlthef/acetalde.html. Accessed May 11th, 2016.

447 FAO (Food and Agriculture Organization of the United Nations). (2003). Chapter 2: Methods of

448 food analysis. FAO food and nutrition paper 77: Food energy methods of analysis and

449 conversion factors. ISBN 92-5-105014-7.

450 http://www.fao.org/docrep/006/y5022e/y5022e03.htm Accessed May 11th, 2016.

451 MSDS. (2008). Material safety data sheet for DMDS. Matheson Tri-Gas.

452 http://www.dogee.org/lab/chemicals/2835.pdf. Accessed May 11th, 2016.

453 USDA (United States Department of Agriculture). (2015). USDA National Nutrient Database for

454 Standard Reference, release 28. http://ndb.nal.usda.gov. Accessed May 11th, 2016.

455 Viala, E. (2008). Water for food, water for life a comprehensive assessment of water

456 management in agriculture. Irrigation Drainage Systems. 22:127-129.

457
458 Table 1. The retention time, standard curves, and published odor threshold values for the
459 primary odorants. H2S: hydrogen sulfide, DMDS: dimethyl disulfide, DMTS: dimethyl
460 trisulfide.

461

Odorant Retention time (min) Selected ion(s) (m/z) Standard curve* Odor th
H2S 2.54 34 y = x /13.4
Acetaldehyde 3.25 44 y = 0.046656 x 0.0078582
Methanethiol 3.48 47 y = x /13.4
Pentanal 8.46 44 &58 y = 0.14963 x 0.11124
DMDS 9.61 94 y = 0.038929 x + 0.00071252
Hexanal 10.76 44 & 56 y = 0.14963 x 0.11124
Benzaldehyde 14.01 105 & 106 y = 0.14963 x 0.11124
DMTS 14.25 126 y = 0.038929 x + 0.00071252
2-Pentyl furan 14.38 81 y = 0.14963 x 0.11124
*
462 Y-axis stands for the peak area of the compound divided by the peak area of ethanethiol; X-axis
463 means quantify of the odorant (ng) in the 15 mL headspace injected.
a
464 The odor threshold values are cited from Ruth, 1986; EPA, 2000; Pohanish, 2012; Verschueren,
465 1983; MSDS, 2008; Rychlik, Schieberle, & Grosch, 1998; Ruth, 1986; Boatright, 2002; and Van
466 Gemert, 2003, respectively.
467 Table 2. Proximate analysis of commercial rice proteins. Values are expressed as Mean
468 Standard deviation. a-dMeans in the same column with different letters are significantly
469 different (P < 0.5).

470

Moisture (%) Protein (%) Fat (%) Ash (%)


RP-G 2.36 0.06c 82.05 0.25b 5.27 0.12a 1.70 0.01b
RP-O 4.56 0.10a 74.64 0.08d 0.04 0.01d 2.31 0.04a
RP-RM 4.17 0.06b 78.84 0.08c 0.54 0.02c 0.96 0.02d
RP-RS1 2.15 0.07d 83.57 0.04a 3.69 0.01b 1.57 0.01c
RP-RS2 2.38 0.04c 83.26 0.40a 3.72 0.13b 1.57 0.01c
471

472
a

b b

a a
d
b b b
b b
c c

473

474

475 Figure 1. Mineral content (ppm) in rice protein samples. a-dMeans of the same mineral with
476 different letters are significantly different (P < 0.5). Error bar indicates standard deviation.
6.00 ab a
bc
5.00
Total sulfite content (ppm)

cd
4.00
d

3.00

2.00

1.00

0.00
RP-G RP-O RP-RM RP-RS1 RP-RS2
477

478

479 Figure 2. Total sulfite content of rice protein samples. a-dMeans with different letters are
480 significantly different (P < 0.5). Error bar indicates standard deviation.

481
482

483

484

0.18
a
0.16 a

0.14 b
Hydroperoxides (mol/g)

0.12

0.1

0.08
c

0.06 c

0.04

0.02

0
RP-G RP-O RP-RM RP-RS1 RP-RS2
485

486

487 Figure 3. Protein hydroperoxides in rice protein samples. a-cMeans of the same mineral
488 with different letters are significantly different (P < 0.5). Error bar indicates standard
489 deviation.
490 Table 3. Mean concentrations of primary rice protein odorants in the headspace of 5%
491 Slurries (mg/m3 in air) by static headspace analysis. Values in parentheses are standard
492 deviations (n = 3). Values in brackets/bold are odor values in air. MT: methanethiol;
493 DMDS: dimethyl disulfide; DMTS: dimethyl trisulfide; n.d.: not detected.
494

Product H2 S Acetaldehyde MT Pentanal DMDS Hexanal Benzaldehyde DMTS

0.350 0.530 1.802 0.110 0.018


RP-G n.d. n.d. n.d. (0.013) (0.045) (0.050) (0.008) (0.001)
[5] [18] [34] [1] [15]

0.434 0.014 0.106 0.340 0.540 0.145 0.021


RP-O n.d. (0.083) (0.006) (0.009) (0.034) (0.042) (0.008) (0.002)
[5] [8] [1] [12] [10] [1] [18]

0.032 0.138 1.575 0.377 5.907 0.756 0.021


RP-RM n.d. (0.036) (0.026) (0.148) (0.024) (0.530) (0.251) (0.003)
[0] [86] [22] [13] [111] [8] [18]

0.057 0.003 0.480 1.522 2.171 0.202 0.042


RP-RS1 n.d. (0.080) (0.004) (0.082) (0.190) (0.290) (0.018) (0.000)
[1] [2] [7] [52] [41] [2] [35]

0.073 0.003 0.509 1.665 2.201 0.232 0.048


RP-RS2 n.d. (0.011) (0.003) (0.004) (0.082) (0.100) (0.012) (0.003)
[1] [2] [7] [57] [42] [2] [40]

495
496
497

27
498 Highlights

499 The primary rice protein odorants were identified using static headspace analysis.

500 Three sulfur-containing compounds were identified among the primary odorants.

501 Acetaldehyde contributed significantly to the odor of RP-O but not other samples.

502 The major lipid-derived odorants included hexanal, pentanal, and 2-pentayl furan.

503 Metals, sulfite, and hydroperoxides were detected in rice protein samples.

504

505

28