Accepted Manuscript

Hydrothermal treatment of rice: Reduction of aflatoxins and bioaccessibility

Marina Nogueira da Silva, Kelly Cristina Massarolo, Larine Kupski, Eliana Badiale
Furlong

PII: S0733-5210(18)30764-1
DOI: https://doi.org/10.1016/j.jcs.2018.12.009
Reference: YJCRS 2688

To appear in: Journal of Cereal Science

Received Date: 3 October 2018

Revised Date: 11 December 2018
Accepted Date: 12 December 2018

Please cite this article as: da Silva, M.N., Massarolo, K.C., Kupski, L., Furlong, E.B., Hydrothermal
treatment of rice: Reduction of aflatoxins and bioaccessibility, Journal of Cereal Science (2019), doi:
https://doi.org/10.1016/j.jcs.2018.12.009.

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.
 1
ACCEPTED MANUSCRIPT

1 Hydrothermal treatment of rice: reduction of aflatoxins and bioaccessibility

2 Marina Nogueira da Silvaa, Kelly Cristina Massaroloa, Larine Kupskia, Eliana Badiale

3 Furlonga*

PT
5

a
6 Laboratório de Micotoxinas e Ciência de Alimentos, Escola de Química e Alimentos,

RI
7 Universidade Federal do Rio Grande - FURG, Avenida Itália km 8, Campus Carreiros, 96203-

SC
8 900 Rio Grande, RS, Brasil.

U
10 Marina Nogueira da Silva: marynogueira29@hotmail.com
AN
11 Kelly Cristina Massarolo: kelly_massa@hotmail.com

12 Larine Kupski: larinekupski@yahoo.com.br

M

13 Eliana Badiale Furlong: dqmebf@furg.br

D

14
TE

15

16
EP

17

18
C

19
AC

20

21

22 *Corresponding author: Eliana Badiale Furlong, Tel: +55 5332336796.

23 Email address: dqmebf@furg.br

 2
ACCEPTED MANUSCRIPT

24 Abstract

25 The reduction and bioaccessibility of aflatoxins B1, B2, G1 and G2 and their relationship with

26 resistant starch in three class of rice after two hydrothermal treatments was studied by

27 Principal Component Analysis (PCA). Microwave oven (84.5%) and electric plate (87.2%)

PT
28 treatments promoted a high level of reduction for all aflatoxins. Bioaccessibility in the range

29 of 58%–100% for aflatoxins B2, G1 and G2 was caused by the microwave treatment and in

RI
30 this treatment the polished rice had a higher bioaccessibility. Hydrothermal treatment

SC
31 efficiently mitigated AFB1 and AFB2 in brown rice. The correlation among aflatoxin

32 bioaccessibility, their reduction during hydrothermal treatment and the level of resistant starch

U
33 was performed by a Principal Component Analysis and it showed no correlation between
AN
34 aflatoxins bioaccessibility and resistant starch, but it showed that the higher reduction of

35 mycotoxin levels the lower its bioaccessibility, which makes this study interesting to
M

36 mitigating human exposure to this contaminant.

D

37
TE

38 Keywords: electric plate; microwave oven; principal component analysis; resistant starch.

39
EP

40

41
C

42
AC

43

44

45

46
 3
ACCEPTED MANUSCRIPT

47 1 Introduction

48 The quality of grains is mainly defined by their nutritional and technological

49 properties, which also determines their economic value. However, it is difficult to apply

50 strategies that are able to mitigate the dangerous effect of fungal contaminants, mainly

PT
51 associated with chronic exposure through the consumption of food that is contaminated with

52 mycotoxins below the maximum acceptable limit (Mishra et al., 2013).

RI
53 Concern for food safety has motivated specific legislation to limit the level of

SC
54 mycotoxins in food and feed (Commission, 2006). Mycotoxins are toxic metabolites produced

55 by toxigenic fungal species that can contaminate grains along the food chain under several

U
56 conditions. These metabolites show large toxicity spectra, low molecular weight, thermal
AN
57 stability and are capable of binding to biomolecules, that masks their levels under analytical

58 determination. Furthermore, they are able to promote damage in several organs (e.g. the liver
M

59 and kidneys) and systems (e.g. digestive, neurologic and dermic) of humans and animals,
D

60 even at low concentrations (Bhat et al., 2010).

TE

61 Aflatoxins (AF) B1, B2, G1 and G2, which are produced by toxigenic species of the

62 genus Aspergillus, stand out because of their frequency in grains and food, thermal stability,
EP

63 occurrence level and carcinogenicity as well as causing other chronic symptoms. Their ability

64 to associate to biopolymers (i.e. starch) has caused concern among researchers because this
C

65 could affect the reliability of determining their presence and could also have biological effects
AC

66 because their acceptable limits are established in raw material and food before cooking.

67 Therefore, the maximum tolerance limits set by government agencies don´t ensure that there

68 are no risks of chronic effects (Van Egmond et al., 2007).

69 Information about AF bioaccessibility is very important to mitigate chronic hazards,

70 mainly regarding foods and feed that are frequently consumed. Rice and its derivatives are

71 consumed worldwide in different forms, such as polished, parboiled, brown, bran and flour,
 4
ACCEPTED MANUSCRIPT

72 and should be evaluated more thoroughly for AF bioaccessibility because of frequent

73 consumption. Although AFs have been found at concentrations below the legal limits in

74 Arabia, Australia, Brazil, China, Egypt and the USA (Al-Zoreky and Saleh, 2017;

75 Katsurayama et al., 2018), it should be highlighted that AF may have associated to the main

PT
76 biopolymer found in these foods, starch, which could have affected their analytical detection.

77 In domestic and industrial processes, rice is hydrothermally treated and the starch

RI
78 becomes gelatinized, and perhaps more bioavailable. Starch digestibility is classified as

SC
79 available or resistant and the proportion of these properties is determined by the grain variety,

80 storage and processing conditions. The available starch is hydrolyzed during digestion, which

U
81 increases glucose, and the resistant form may be fermentable in human intestines by
AN
82 microorganisms or eliminated as indigestible waste (Frei et al., 2003).

83 The effect of hydrothermal actions on aflatoxin reduction has been reported in the
M

84 literature, as in the work of Hussain and Luttfullah (2009), where there was a reduction of
D

85 72.5% in AFB1 during microwave treatment of Basmati rice. Despite the importance of the
TE

86 study of processes to reduce aflatoxin levels, it is also necessary to study the bioaccessibility

87 of these compounds after heat treatment because if AFs associate to resistant starch they are
EP

88 not available for intestinal absorption.

89 Based on this, in this study we evaluate the bioaccessibility of AF B1, B2, G1 and G2 in
C

90 polished, parboiled and brown rice and their relationship with resistant starch after
AC

91 hydrothermal treatment by Principal Component Analysis (PCA). The aim of this study was

92 submitted the rice to hydrothermal treatments usually used in a domestic cooking, such as

93 electric plate and microwave oven, then evaluate which treatment would cause lees exposure

94 to aflatoxin.

95
 5
ACCEPTED MANUSCRIPT

96 2 Materials and Methods

97 2.1 Sample preparation

98 Rice (Oryza sativa L.) samples of type 1, class long and thin (brown, parboiled and

99 polished) were purchased from market of the South Brazilian. It was obtained the three rice

PT
100 types for each brand (3 brands: low, medium and high cost). All brands for each rice type

101 were homogenized and stored in dry conditions at 25 ºC, getting a sample for each type of

RI
102 rice. Samples were ground to a particle size less than 0.5 mm (35 mesh) for determination of

SC
103 chemical composition and contamination by AFs.

104

U
105 2.2 Chemical composition
AN
106 We determined the centesimal composition of the raw samples. Moisture, ash, protein

107 and lipids were assessed according AOAC (2000) methods, numbers 935.29; 923.03, 920.87,
M

108 920.85, respectively.

D

109 Amylose content was determined using colorimetric methods (Martinéz and Cuevas,
TE

110 1989). Rice samples (100 mg) were weighed and transferred to volumetric flasks, then we

111 added 1 ml of 96% ethanol and 9 mL of 1M NaOH to the samples. After 16 h, 5 mL of the
EP

112 mixture was transferred to a volumetric flask, then we added 1 mL of 1M acetic acid, 2 mL of

113 2% (p/v) iodine solution and filled the flask with distilled water. The transmittance was
C

114 determined after 30 minutes at 610 nm. The amylose standard curve ranged from 0.4 to 2
AC

115 mg/mL. The total starch content was determined using iodometry and amylopectin was

116 estimated as the difference of total starch and amylose.

117

118 2.3 Hydrothermal treatment of rice

119 In 20 g of rice (brown, parboiled and polished) we added 0.2 g of NaCl and 0.4 g of

120 soybean oil and water (1:2 p/v) and submitted the mixture to hydrothermal treatment in
 6
ACCEPTED MANUSCRIPT

121 microwave ovens (manufacturer Brastemp, model BMS35BBBNA, tension 220V, useful

122 power 820W, network frequency 60Hz, operating frequency 2450MHz, key thermal breaker

123 10A, consumption in microwave mode 1440W, permissible voltage fluctuations 198~242,

124 cavity volume 30L, external product dimension (height x width x depth) 300 x 539 x 420) or

PT
125 electric plates (manufacturer Velp Scientifica, model ARE heating magnetic stirrer, tension

126 115V, network frequency 60Hz, construction material aluminum frame with epoxy paint,

RI
127 heating plate aluminum alloy with special coating, diameter of the plate 155mm, power

SC
128 630W, dimensions 165x115x280mm, temperature adjustment up to 370 °C) until it obtained

129 the textural characteristics of cooked rice. The rice was considered cooked when the product

U
130 didn’t have lumps or aggregates and was approximately 2–3 times the bulk volume of the raw
AN
131 volume that was initially used. The complete gelatinization of the rice was tested by pressing

132 the cooked grain between two glass plates until the grain center did not possess a “hard
M

133 center”, which was evaluated every two minutes after the water began to boil (Juliano, 1985).
D

134 Supplement 1 shows the parameters that were adopted for each rice under thermal
TE

135 treatments.

136 To define the parameters for efficiently cooking rice, we determined the texture
EP

137 parameters with a texturometer (TA XT2, software Exponent-Stable Micro System). We used

138 5 g in a cell that compressed 80% of the cooked grain twice with a test velocity of 0.5 mm.s-1
C

139 and a distance of 20 mm. The following parameters were determined: toughness,
AC

140 adhesiveness, gumminess, chewiness and elasticity.

141 Before chemical determination, the samples that were hydrothermally treated were

142 kneaded and sifted in a sieve to reduce them to 0.5 mm (35 mesh) and we analyzed them

143 according to the details in item 2.2.

144
 7
ACCEPTED MANUSCRIPT

145 2.4 Available and resistant starch

146 We determined the amount of available and resistant starch according to the protocol

147 described by AOAC (2000) number 996.11 in rice samples immediately after cooking and

148 after 48 h of refrigeration (10°C). After enzymatic hydrolysis, we quantified the glucose

PT
149 released from the starch using 3.5 dinitrosalicyclic colorimetric reactions (Miller, 1959), and

150 we adopted a conversion factor of 0.9 to convert glucose to starch.

RI
151

SC
152 2.5 Reduction of AFLAs by hydrothermical treatment

153 The aflatoxin levels of the rice sample were determined and the samples aren´t

U
154 naturally contaminated, so it was necessary to spike them to evaluate the effect of the two
AN
155 hydrothermal treatments in the reduction of aflatoxins. For this, each type of rice was spiked

156 before the hydrothermal treatment with aflatoxins in concentrations equal to 10 fold the limit
M

157 of quantification of the method.

D

158 AFLA extraction was performed according to Rubert et al. (2011) with modifications.
TE

159 To assess aflatoxins levels before the thermal treatment, aliquots of 1 g of “in nature” rice

160 were transferred to a mortar (30 mL capacity), gently homogenized with a pestle with 25 mg
EP

161 of C18 and 25 mg of Na2SO4 for 5 min to obtain a homogeneous mixture. The mixture was

162 poured into a centrifuge polypropylene tube to which we added 10 mL of

C

163 acetonitrile:methanol (50:50, v/v) and the content was thoroughly agitated in a vortex for 3
AC

164 min. The tubes were centrifuged at 3220 xg for 10 min, then the extract was collected, dried at

165 60 °C, dissolved in 1000 µL ultrapure water and acetonitrile (90:10, v/v) and injected into the

166 HPLC-FD under established conditions. To assess aflatoxin levels after the thermal treatment,

167 the same procedure described above was conducted, using 3 g of cooked rice.
 8
ACCEPTED MANUSCRIPT

168 The reduction of AFLAs levels by the thermal treatments were calculated correlating

169 AFLAs concentration on cooked rice (after thermal treatment) with their concentration on “in

170 nature” rice (before thermal treatment).

171

PT
172 2.6 Bioaccessibility of AFLAs

173 The bioaccessibility of cooked rice was determined with the method developed and

RI
174 validated by the National Institute of Public Health and Environmental (RIVM, Bilthoven, the

SC
175 Netherlands).The in vitro digestion model was validated comparing the effects of adsorbent

176 materials on the bioaccessibility of aflatoxin B1 and ochratoxin A from food with in vivo data

U
177 in experimental animals. In 5 out of 6 combinations of adsorbents and mycotoxins, the effects
AN
178 of the adsorbents on the bioaccessibility of aflatoxin B1 and ochratoxin A in the in vitro

179 digestion model were in agreement with the effects observed in vivo. None of the effects with
M

180 the in vitro digestion model disagreed with the effects observed in vivo. This method
D

181 considers the three main steps of human digestion (mouth, stomach and intestines)
TE

182 (Versantvoort et al., 2005). Solutions of saliva, gastric juice and biliary juice were prepared

183 according to Kabak and Ozbey (2012).

EP

184 The hydrothermal treatment promoted a high reduction of aflatoxins, then the aflatoxin

185 spiked was realized after the hydrothermal treatment in concentration equal to 10 fold the
C

186 limit of the method and then submitted to the bioaccessibility method. Into a polypropylene
AC

187 tube we added 4.5 g of hydrothermally spiked cooked rice (brown, parboiled and polished)

188 and 6 mL of saliva (pH: 6.5) and incubated the mixture for five minutes at 37 °C. Next, 12

189 mL of gastric juice (pH 2.5) was added to the sample and was homogenized for 2 h at 37 °C

190 in an orbital shaker at 150 rpm. Finally, 12 mL of duodenal juice (pH 6.5), 6 mL of biliary

191 juice and 2 mL of a NaHCO3 1M were added and homogenized for 2 h at 37 °C in an orbital

192 shaker at 150 rpm. At the end of the in vitro digestion process, the digestion tubes were
 9
ACCEPTED MANUSCRIPT

193 centrifuged for five minutes at 3220 xg, which yielded the chyme (supernatant) in which the

194 concentration of aflatoxins was determined and the digested matrix (the pellet).

195 AFLAs were extracted from 20 mL of the chyme with 5 mL of chloroform (three

196 times) and the extract was evaporated under a N2 stream at 60°C (Kabak and Ozbey, 2012).

PT
197 Mycotoxins were determined by HPLC-FD. The bioaccessibility was estimated as a

198 percentage, which was calculated as the relation between the mycotoxin concentration in the

RI
199 supernatant and the concentration before the digestion steps.

SC
200 2.7 Determination of AFLAs

201 AF standards were dissolved in 1 mL of ultrapure water and acetonitrile (90:10, v/v)

U
202 and we injected 20 µL into a HPLC system coupled to a fluorescence detector equipped with
AN
TM
203 a post-column photochemical derivatization (RomerDerivatization Unit RDU ), which

204 excites the analytes with 254 nm UV light.

M

205 A chromatographic elution with an analytical column Kromasil C18 (5µm, 150 x 4.6
D

206 mm) was performed at 40 ºC using a mobile phase of ultrapure water:acetonitrile:methanol

(62:12:26, v/v/v) at a flow rate of 1.0 mL·min-1 and excitation and emission wavelengths were
TE

207

208 set at 365 and 440 nm, respectively.

EP

209 The limit of detection (LOD) and the limit of quantification (LOQ) of the method for

210 each AFLA were considered three and ten times the ratio of signal to baseline (noise),
C

211 respectively. The linearity was evaluated through standard calibration curves in the
AC

212 concentration range of the LOQ of each AFLA to a concentration equivalent to 100-fold LOQ

213 value.

214 The accuracy of the method was evaluated regarding the recovery assays, in

215 compliance with SANTE/11945/2015 (SANTE 2016) and ANVISA (2003). Aliquots of 3 g

216 of cooked rice and 1 g of “in nature” were spiked with standard AFLAs at three levels for

217 each AFLA. The levels of fortification were concentrations equivalent to the LOQ, 5-fold
 10
ACCEPTED MANUSCRIPT

218 LOQ and 10-fold LOQ. Each fortification level was extracted in triplicate and injected three

219 times (n = 9). The precision of the method was evaluated regarding the repeatability, with

220 nine determinations. The extraction of the sample by MSPD was performed at three different

221 fortification levels, in triplicate.

PT
222

223 2.8 Statistical analysis

RI
224 All assays were performed in triplicate. The chemical composition was analyzed using

SC
225 an ANOVA of the average of the triplicate assays, followed by a Tukey's test at a 5% level

226 (p<0.05) using the program "Statistic" (version 7.0, StatSoft, Inc., Tulsa, USA). Texture

U
227 attributes, available and resistant starch, aflatoxin reduction and bioaccessibility were
AN
228 analyzed by Factorial ANOVAs, using treatment and rice class as factors, followed by

229 Tukey's tests at a 5% level (p < 0.05). The multivariate analysis was performed using PAST
M

230 software (folk.uio.no/ohammer/past). A principal component analysis (PCA) was also applied
D

231 to the data set (for resistant and available starch, AFLAs reduction and bioaccessibility).
TE

232

233 3 Results and Discussion

EP

234 Samples of commercial rice that are processed differently are compounds of grains

235 from different varieties that are recommended for cultivation in southern Brazil because of
C

236 their productivity and adaptability to the region's environmental conditions. In this study, we
AC

237 determined the percentages of macromolecules and ratio of amylose to amylopectin in several

238 types of rice (Supplement 2).

239 We found similar rice compositions to what was found in several previous studies for

240 rice samples from the class long and thin. The difference among them was determined using

241 the benefit process (NEPA - Núcleo de Estudos e Pesquisas em Alimentação, 2011; Walter et

242 al., 2005). Our samples showed this behavior (Supplement 2).
 11
ACCEPTED MANUSCRIPT

243 The main component of rice is starch, which is the same for all class samples,

244 suggesting that they have similar genetic information and that they were exposed to the same

245 environmental conditions (Frei et al., 2003). Rice can be classified according to the content of

246 amylose as waxy (1–4%), low content of amylose (12–19%), intermediary (20–24%) or high

PT
247 content (23–32%); however, in terms of amylose content our samples are classified as

248 intermediary. This characteristic is technologically important because there is a direct relation

RI
249 between the amylose content and resistant starch potential, which affects the food digestibility

SC
250 and the properties of the product’s formulation.

251 The ratio between amylose and amylopectin were 0.31, 0.44 and 0.36 for brown,

U
252 parboiled and polished rice, respectively. It is important to mention that amylopectin
AN
253 determines the crystallinity of the rice grain and the gelatinization temperature because it

254 promotes structural stability of the granule as a consequence of resistance to gelatinization.

M

255 The intermediate amylose content of the samples suggests a melting point that is reduced in
D

256 the crystal regions; however, the amylopectin chain needs more energy to dissociate (Singh et
TE

257 al., 2003).

258
EP

259 3.1 Effects of hydrothermal treatment on the rice texture profile

260 The water volume and cooking time of the rice samples were defined by the
C

261 transparency of the grain when pressed between glass plates. Rice that was cooked using
AC

262 microwaves required a higher water volume than rice cooked on electric plates

263 (Supplement 1) because the evaporation was greater during the treatment as a consequence

264 of the friction promoted by the rotation of dipolar molecules in the electric field. Each rice

265 class needed a specific water volume for cooking; the brownand parboiled required more

266 water than the polished because they possess outer layers (pericarp, aleurone and integument)

267 that make water diffusion into the inner layers difficult. Also, the parboiling process made the
 12
ACCEPTED MANUSCRIPT

268 structure more rigid as a consequence of amylose and amylopectin chain rearrangement

269 (Mohapatra and Bal, 2006).

270 Using microwaves, the cooking time was shorter because the energy distribution was

271 more efficient than using a conventional electric plate, and this treatment can´t guarantee heat

PT
272 homogeneity. Brown rice required more time (approximately 1.5 times) to cook under both

273 treatments, which, as stated above, was due to the outer layers making energy and mass

RI
274 transference difficult.

SC
275 The plate test to define cooked rice is not quantitative, so the samples were evaluated

276 using the following controls for the cooking parameters toughness, adhesiveness, gumminess,

U
277 chewiness and elasticity in a texturometer (Table 1). The parameters that are used to define a
AN
278 food’s texture profile in a texturometer are based on sensorial human perception; however,

279 their determination is carried out under forced conditions that were previously established to
M

280 guarantee the reliability of the measure. Toughness measures the maximum force needed to
D

281 compress the sample into a previously established condition, and this measure is correlated
TE

282 significantly with the gelatinization grade (Swasdisevi et al., 2010). Therefore this value in

283 the treated rice samples was adopted as a control indicator of the rice reaching the point where
EP

284 it was considered cooked. Adhesiveness indicates the force that is required to remove food

285 that adheres to the tongue, teeth and mucosa. Gumminess is defined as the energy that is
C

286 needed to disintegrate a food into a pasty state. Chewiness is defined as the number of chews
AC

287 needed to make a food doughy. Elasticity is defined as the ability of a food to return to its

288 original form after compression. All of these measures are realized under established force,

289 and they are also related to the gelatinization grade, but they are less directly associated with

290 this property. The parboiled samples submitted to both treatments stand out in relation to their

291 texture parameters, which may be caused by major gelatinization changes that are promoted

292 by water logging. There is little information regarding texture profile of cooked rice, but
 13
ACCEPTED MANUSCRIPT

293 similar values were found by (Villanova et al., 2014) for brown and parboiled rice after

294 cooking rice on electric plates (toughness 54.61 and 56.78 N). There was no information

295 about other texture parameters or rice cooked under microwaves until this study.

296

PT
297 3.2 Available and resistant starch in cooked rice

298 Starch fractions in the rice samples that were cooked in microwave ovens and on

RI
299 electric plates were determined immediately after treatment and after 48 h at 10 ºC (Table 2).

SC
300 The source of energy to cook the rice samples, the rice class and the cooking time

301 significantly affected (p < 0.05) the level of resistant starch. The polished rice sample stands

U
302 out regarding resistant starch when cooked on an electric plate (138.1 mg·g-1) and after 48 h at
AN
303 10 ºC the resistant starch content reduced 19%, but when the rice was cooked in a microwave

304 oven it caused an increase (41%) in the resistant starch content. The major starch
M

305 retrogradation was promoted by the electromagnetic field on the starch chain under
D

306 microwaves and the gelatinization grade was higher immediately after the treatment.
TE

307 However, under refrigeration the starch chains reorganized, forming crystalline regions that

308 were more resistant to enzymatic hydrolysis (resistant starch) (Incropera and Dewitt, 2014).
EP

309 In general, the rice samples cooked on an electric plate showed a reduction in resistant

310 starch and an increase when it was cooked in a microwave oven. This behavior reinforces the
C

311 idea that microwave electromagnets promote starch retrogradation after cooling. When
AC

312 gelatinized rice was submitted to the cooling, the starch molecules lost energy and the

313 hydrogen bonds were stronger and more organized, leading to the formation of simple and

314 double helices and crystalline areas, which were less digestible, called resistant starch type 3.

315 The formation of resistant starch type 3 is affected by many factors such as storage

316 temperature and time, starch source, association with other molecules and process parameters

317 (Rosin et al., 2002).

 14
ACCEPTED MANUSCRIPT

318 In our experiments the brown and parboiled samples differed significantly (p < 0.05)

319 in regard to their resistant starch. This could be due to the fact that in brown grains the

320 resistant starch that is resistant to hydrolysis by the amylases is class 2 and in parboiled rice it

321 is class 3 as in processed food where the starch structure is modified by temperature. In both,

PT
322 the digestion is slower than in resistant starch class 1 (Casiraghi et al., 1993). Chung et al.

323 (2009) found highly resistant starch in samples with high amylose content (e.g. in peas and

RI
324 lentils) and the same relationship was founded by Helbig et al. (2008) in rice that was waxy

SC
325 (31.6%), intermediary (21.8%) and had a low (6.3%) percentage of amylose.

326

U
327 3.3 Aflatoxin levels after hydrothermical treatments
AN
328

329 The limits of quantification for AFB1, AFB2, AFG1 and AFG2 were 0.12, 0.02, 0.04
M

330 and 0.1 ng g-1, respectively. It is important to emphasize that it is unusual to validate
D

331 analytical methods for aflatoxin determination in cooked samples. However, to ensure the
TE

332 reliability of our results we validated the method previously used for raw samples for cooked

333 samples (Table 3) because the gelatinization could affect the recovery and the repeatability.
EP

334 There was a trend of improving the analytical parameters in the cooked samples; therefore the

335 method of determining aflatoxins in rice samples submitted to the digestion process
C

336 satisfactorily showed them in the chyme.

AC

337 Aflatoxins are thermal-resistant compounds, making it difficult to mitigate their

338 presence in feed and foods by conventional processes that apply moderated conditions such as

339 cooking, extrusion and baking. However, evaluating their effects is important for determining

340 acceptable concentrations of them in commodities and foods (Pankaj et al., 2018). Under the

341 hydrothermal treatment conditions that were used in this study, the percentage of reduction

342 was estimated (Table 4) as the level of each mycotoxin (Supplement 3) in the samples before
 15
ACCEPTED MANUSCRIPT

343 and after cooking. The significant difference among the hydrothermal treatments and rice

344 classes for aflatoxin reductions were determined by factorial analyses of variance (95%).

345 The effect of the type of hydrothermal treatment was only significant (p < 0.05) for

346 aflatoxin B2 reduction. In general, there was a high level of reduction for all aflatoxins, with

PT
347 average values of 84.5% for rice treated with microwave ovens and 87.2% for those treated

348 with electric plates. Other research showed a decrease in aflatoxin reduction when the electric

RI
349 plate treatment was applied, and it was affected by parameters such as dry or moist, fast and

SC
350 short time and source of energy (electric or electromagnetic). However, there was no

351 consistency among the results (Hussain and Luttfullah, 2009; Pankaj et al., 2018).

U
352 In addition to our satisfactory results of decreasing aflatoxins, it is also important to
AN
353 evaluate the amount of aflatoxins that would be available for absorption after digestion, so we

354 determined the amount of aflatoxins in chyme (intestinal content) from a simulated digestive
M

355 process
D

356
TE

357 3.4. Aflatoxin bioaccessibility in cooked rice

358
EP

359 The aflatoxin bioaccessibility was determined in cooked rice (Table 5 and

360 Supplement 4), for this after the hydrothermal treatment the rice cooked was spiked with
C

361 aflatoxins in concentrations equal to 10 fold the limit of quantification of the method, because
AC

362 in this way the initial concentration of aflatoxin would be assured and known.

363 The type of thermal treatment had a significant effect for AFLAs B2, G1 and G2, and

364 the microwave treatment was responsible for the greater bioaccessibility of aflatoxins in the

365 range of 58%–100%. Regarding the rice class, there was a significant difference (p < 0.05) for

366 all aflatoxins, and in general the polished grains had a higher bioaccessibility, which may be

367 associated with the higher resistant starch content in this class.
 16
ACCEPTED MANUSCRIPT

368 Therefore, the hydrothermal treatment efficiently mitigated AFB1 and AFB2 in brown

369 rice, although it has a lower resistant starch content. However, the presence of lipids, cellulose

370 and lignin may also contribute to the decrease the aflatoxin bioaccessibility. The decrease of

371 aflatoxin bioaccessibility in parboiled rice may be due to the presence of resistant starch type

PT
372 3 that is produced during cooking.

373 These results showed that the hydrothermal treatment promotes the association of

RI
374 aflatoxins and other macromolecules of the matrix such as resistant starch and others. The

SC
375 interaction may decrease the recovery of them during analytical determination,

376 underestimating the risk of exposure.

U
377 To better understand the relationship between aflatoxin bioaccessibility, their
AN
378 reduction during hydrothermal treatment and the level of resistant starch, we performed a

379 Principal Components Analysis (Figure 1). During the PCA analysis two regions were
M

380 highlighted. The circle encompasses the resistant starch variables, bioaccessibility of AFB1
D

381 and reduction of AFG1. A Pearson's correlation analysis shows a positive and significant
TE

382 relationship between the reduction of AFG1 and the bioaccessibility of AFB1 (R: 0.76, p =

383 0.08), indicating that the greater the reduction of AFG1 content, the greater the
EP

384 bioaccessibility of AFB1.

385 The rectangle encompasses the variables of available starch (48 h) and the
C

386 reductions of AFLAs B1 and G2. According to the Pearson's correlation for these variables, a
AC

387 positive and significant correlation between the available starch content and the reduction of

388 AFB1 (R = 0.77, p = 0.07) and AFG2 (R = 0.71, p = 0.11) was observed, indicating that for

389 reduction during the hydrothermal process it would be interesting to apply a treatment that

390 significantly increases the available starch content for these mycotoxins (AFB1 and AFG2),

391 as occurs during treatment on an electric plate in the present work. The opposite behavior is

392 found to reduce AFG1, indicated by the angle of 180° with the variable AD 48 h (Figure 1),
 17
ACCEPTED MANUSCRIPT

393 thus presenting a negative and significant correlation (R = -0.751; p = 0.09). Therefore, for

394 the reduction of AFG1, it would be more interesting to use the microwave, which presents

395 lower levels of AD 48 h, compared to the treatment on the electric plate.

396 The negative and significant correlation between the reduction of AFB2 and its

PT
397 bioaccessibility (R = -0.95, p = 0.003), indicated in the graph by the angle of 180° between

398 the two variables, should also be highlighted. This indicates that the higher the reduction of

RI
399 mycotoxin levels, the lower its bioaccessibility, which makes this study interesting from the

SC
400 point of view of mitigating human exposure to this contaminant. Therefore, the correlation

401 between the reduction caused by the thermal treatment of this mycotoxin and the reduction of

U
402 its impact on human health was verified.
AN
403

404 4. Conclusion
M

405 The two hydrothermal treatments (microwaves and electric plate) promote apparent

406 reduction of the studied mycotoxins (above 84%). The microwave treatment was responsible
D

407 for the greater bioaccessibility of aflatoxins in the range of 58%–100% and the polished rice
TE

408 had a higher bioaccessibility, which was associated with the higher resistant starch content.
EP

409 Principal Components Analysis showed no correlation between aflatoxins bioaccessibility and

410 resistant starch, but it showed that the higher reduction of mycotoxin levels the lower its
C

411 bioaccessibility.
AC

412

413 Acknowledgments

414 The authors acknowledge the support of Coordenação de Aperfeiçoamento de Pessoal de

415 Nível Superior (CAPES, Brazil), Conselho Nacional de Desenvolvimento Científico e

416 Tecnológico (CNPq, Brazil) and Fundação de Amparo à Pesquisa do Estado do Rio Grande
 18
ACCEPTED MANUSCRIPT

417 do Sul (FAPERGS, Brazil). L. Kupski received a postdoctoral fellowship by CNPq

418 (150439/2017-2).

419

420 Funding: This study was financed in part by the Coordenação de Aperfeiçoamento de

PT
421 Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

422

RI
423 Compliance with Ethics Requirements

SC
424 Conflict of Interest: Marina Nogueira da Silva declares that she has no conflict of interest.

425 Kelly Cristina Massarolo declares that she has no conflict of interest. Larine Kupski declares

U
426 that she has no conflict of interest. Eliana Badiale Furlong declares that she has no conflict of
AN
427 interest.

428 Ethical Approval: This article does not contain any studies with human participants or
M

429 animals performed by any of the authors.

D

430 Informed Consent: Not applicable.

TE

431

432 References
EP

433 Al-Zoreky, N.S., Saleh, F.A., 2017. Limited survey on aflatoxin contamination in rice. Saudi

434 Journal of Biological Sciences. https://doi.org/10.1016/j.sjbs.2017.05.010

C

435 ANVISA, 2003. Guía para validação de métodos analíticos e bioanalíticos. Resolução RE n
AC

436 899, 29 de maio de 2003 1–15. http://portal.anvisa.gov.br/documents/10181/2718376/

437 RE_899_2003_COMP.pdf/ff6fdc6b-3ad1-4d0f-9af2-3625422e6f4b

438 AOAC, 2000. Official Methods of Analysis of AOAC International. Association of Official

439 Analysis Chemists International Method ce 2-66.

440 https://doi.org/10.3109/15563657608988149
 19
ACCEPTED MANUSCRIPT

441 Bhat, R., Rai, R. V, Karim, A.A., 2010. Mycotoxins in Food and Feed : Present Status and

442 Future Concerns. Comprehensive Reviews in Food Science and Food Safety 9, 57–81.

443 Casiraghi, M.C., Brighenti, F., Pellengrini, N., Leopardi, E., Testolin, G., 1993. Effect of

444 processing on rice starch digestibiliby evaluated by in vivo and in vitro methods. Journal

PT
445 of Cereal Science 17, 147–156.

RI
446 Chung, H.J., Liu, Q., Hoover, R., 2009. Impact of annealing and heat-moisture treatment on

447 rapidly digestible, slowly digestible and resistant starch levels in native and gelatinized

SC
448 corn, pea and lentil starches. Carbohydrate Polymers 75, 436–447.

U
449 https://doi.org/10.1016/j.carbpol.2008.08.006
AN
450 Commission, E., 2006. Commission regulation (EC) 401/2006 of 23 february 2006 laying

451 down the methods of sampling and analysis for the official control of the levels of
M

452 mycotoxins in foodstuffs. Official Journal of the European Union 2006, 5–24.
D

453 Frei, M., Siddhuraju, P., Becker, K., 2003. Studies on the in vitro starch digestibility and the
TE

454 glycemic index of six different indigenous rice cultivars from the Philippines. Food

455 Chemistry 83, 395–402. https://doi.org/10.1016/S0308-8146(03)00101-8

EP

456 Helbig, E.., Dias, A.R.G.., Tavares, R.A.., Shirmer, M.A.., Elias, M.C., 2008. Arroz
C

457 parboilizado: efeito na glicemia de ratos wistar. Archivos Latinoamericanos de Nutricíon

AC

458 58, 149–155.

459 Hussain, A., Luttfullah, G., 2009. Reduction of Aflatoxin-B1 and Ochratoxin-A levels in

460 Polished Basmati Rice (Oryza sativa Linn.) by Different Cooking Methods. Journal of

461 the Chemical Society of Pakistan 31, 911–915.

462 Incropera, F.P., Dewitt, D.P., 2014. Fundamentos de transferência de calor e massa, sixth ed.
 20
ACCEPTED MANUSCRIPT

463 Ltc, Rio de Janeiro.

464 Juliano, B.O., 1985. Rice: chemistry and technology, second ed. American Association of

465 Cereal Chemists, Saint Paul.

466 Kabak, B., Ozbey, F., 2012. Assessment of the bioaccessibility of aflatoxins from various

PT
467 food matrices using an in vitro digestion model and the efficacy of probiotic bacteria in

RI
468 reducing bioaccessibility. Journal of Food Composition and Analysis 27, 21–31.

469 https://doi.org/10.1016/j.jfca.2012.04.006

SC
470 Katsurayama, A.M., Martins, L.M., Iamanaka, B.T., Fungaro, M.H.P., Silva, J.J., Frisvad,

U
471 J.C., Pitt, J.I., Taniwaki, M.H., 2018. Occurrence of Aspergillus section Flavi and
AN
472 aflatoxins in Brazilian rice: From field to market. International Journal of Food

473 Microbiology 266, 213–221. https://doi.org/10.1016/j.ijfoodmicro.2017.12.008

M

474 Martinéz, C., Cuevas, F., 1989. Evaluacion de la calidad culinaria y molinera del arroz, third
D

475 ed. CIAT, Cali.

TE

476 Miller, 1959. Use of DinitrosaIicyIic Acid Reagent for Determination of Reducing Sugar.
EP

477 Analytical Chemistry 31, 426–428. https://doi.org/10.1021/ac60147a030

478 Mishra, S., Ansari, K.M., Dwivedi, P.D., Pandey, H.P., Das, M., 2013. Occurrence of
C

479 deoxynivalenol in cereals and exposure risk assessment in Indian population. Food
AC

480 Control 30, 549–555. https://doi.org/10.1016/j.foodcont.2012.07.041

481 Mohapatra, D., Bal, S., 2006. Cooking quality and instrumental textural attributes of cooked

482 rice for different milling fractions. Journal of Food Engineering 73, 253–259.

483 https://doi.org/10.1016/j.jfoodeng.2005.01.028

484 NEPA - Núcleo de Estudos e Pesquisas em Alimentação, 2011. Tabela brasileira de

 21
ACCEPTED MANUSCRIPT

485 composição de alimentos. NEPA - Unicamp 161. https://doi.org/10.1007/s10298-005-

486 0086-x

487 Pankaj, S.K., Shi, H., Keener, K.M., 2018. A review of novel physical and chemical

488 decontamination technologies for aflatoxin in food. Trends in Food Science and

PT
489 Technology 71, 73–83. https://doi.org/10.1016/j.tifs.2017.11.007

RI
490 Rosin, P.M., Lajolo, F.M., Menezes, E.W., 2002. Measurement and characterization of

491 dietary starches. Journal of Food Composition and Analysis 15, 367–377.

SC
492 https://doi.org/10.1006/jfca.2002.1084

U
493 Rubert, J., Soler, C., Mañes, J., 2011. Evaluation of matrix solid-phase dispersion (MSPD)
AN
494 extraction for multi-mycotoxin determination in different flours using LC-MS/MS.

495 Talanta 85, 206–215. https://doi.org/10.1016/j.talanta.2011.03.046

M

496 SANTE, 2016. Analytical Quality Control and Method Validation Procedures for Pesticide
D

497 Residues Analysis. European Commission 42. https://ec.europa.eu/food/sites/food

TE

498 /files/plant/docs/pesticides_mrl_guidelines_wrkdoc_2017-11813.pdf
EP

499 Singh, N., Singh, J., Kaur, L., Sodhi, N.S., Gill, B.S., 2003. Morphological, thermal and

500 rheological properties of starches from different botanical sources. Food Chemistry 81,
C

501 219–231. https://doi.org/10.1016/S0308-8146(02)00416-8

AC

502 Swasdisevi, T., Sriariyakula, W., Tia, W., Soponronnarit, S., 2010. Effect of pre-steaming on

503 production of partially-parboiled rice using hot-air fluidization technique. Journal of

504 Food Engineering 96, 455–462. https://doi.org/10.1016/j.jfoodeng.2009.08.026

505 Van Egmond, H.P., Schothorst, R.C., Jonker, M.A., 2007. Regulations relating to mycotoxins

506 in food : Perspectives in a global and European context. Analytical and Bioanalytical
 22
ACCEPTED MANUSCRIPT

507 Chemistry 389, 147–157. https://doi.org/10.1007/s00216-007-1317-9

508 Versantvoort, C.H.M., Oomen, A.G., Van De Kamp, E., Rompelberg, C.J.M., Sips, A.J.A.M.,

509 2005. Applicability of an in vitro digestion model in assessing the bioaccessibility of

510 mycotoxins from food. Food and Chemical Toxicology 43, 31–40.

PT
511 https://doi.org/10.1016/j.fct.2004.08.007

RI
512 Villanova, F.A., Antunes, M.D., Bubolz, V., Schwartz, J.Á., Tadeu, R., 2014. Parâmetros

513 Viscoamilográficos e de Cocção de Arroz Integral , Parboilizado Integral , Preto e

SC
514 Vermelho Após o Beneficiamento. Abrapos, 684–691.

U
515 Walter, M., Silva, L.P. Da, Emanuelli, T., 2005. Amido resistente: características físico-
AN
516 químicas, propriedades fisiológicas e metodologias de quantificação. Ciência Rural 35,

517 974–980. https://doi.org/10.1590/S0103-84782005000400041

M

518
D

519
TE

520
C EP
AC
 ACCEPTED MANUSCRIPT
Figure captions

Fig. 1 - Principal Components Analysis for the variables under studied

PT
RI
U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
1 Table 1 – Texture profile of whole, parboiled and polished cooked rice
Thermal Toughness Adhesiveness Gumminess Chewiness Elasticity
Rice
source (N) (N.s-1) (N) (N.mm-1) (mm)
Brown 95.10b(4.4) -0.73c (18.0) 18.20c (13.2) 9.96b (12.8) 0.07b (11.9)
Microwaves Parboiled 136.14a(10.9) -1.12c (7.2) 36.42a (2.0) 26.21a (18.0) 0.10a (14.0)
Polished 131.75a(12.2) -3.19b (5.4) 31.62b (1.3) 8.40b (4.1) 0.10a (14.7)

PT
Brown 54.94cd(1.9) -4.17a (3.6) 8.19d (13.8) 1.29c (3.5) 0.05bc (12.0)
Electric plate Parboiled 66.61c(5.4) -2.93b (17.4) 10.65d (2.9) 5.20bc (1.4) 0.06b (13.9)
32.67d (17.3) -4.83a (13.3) 3.61e (13.3) 0.18c (0.1) 0.03c (15.1)

RI
Polished
2 Different letters indicate significant difference (p<0.05) for factorial analysis of
3 treatment*class.

U SC
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
4 Table 2 – Available and resistant starch in rice hydrothermal treated
0 h (after cooking) 48 h (10 ºC)
Thermal Available
Rice class Resistant Resistant Available
source -1
Starch (mg.g- -1
Starch (mg.g ) 1
Starch (mg.g ) Starch (mg.g-1)
)

Brown 61.6d (4.9) 569.4ab (1.9) 72.9c (2.5) 662.9a (2.5)

Microwaves Parboiled 71.8c (5.8) 519.0c (1.6) 84.7b (5.8) 550.1c (2.1)

PT
Polished 84.9b (6.0) 539.2bc (3.1) 119.5a (3.1) 550.1c (2.9)

81.7bc(6.8) 598.4a (3.1) 65.1c (5.4) 583.8bc (2.2)

RI
Brown

Electric plate Parboiled 83.1bc(5.2) 599.2a (2.8) 76.4bc (9.4) 648.0a (1.9)

SC
Polished 138.1a(3,6) 580.2ab (3.6) 111.8a (1.5) 600.9b (1.3)

5 Different letters indicate significant difference (p<0.05) for factorial analysis of

6 treatment*class.

U
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
7 Table 3 – Aflatoxins recoveries on “in nature” and cooked rice
Recoveries (%)
Raw samples Hydrothermal treated
Brown rice
AFB1 AFB2 AFG1 AFG2 AFB1 AFB2 AFG1 AFG2
LOQ 60.3 (7.4) 70.3 (8.2) 73.2 (9.3) 109.7 (15.3) 65.8 (9.4) 78.4 (17.8) 68.3 (10.3) 74.5 (4.6)

PT
5LOQ 65.6 (12.3) 99.8 (11.7) 103.3 (6.9) 101.9 (16.7) 75.9 (8.2) 72.6 (15.6) 79.9 (11.1) 112.0 (13.4)
10LOQ 66.4 (15.7) 86.0 (3.6) 60.8 (7.8) 82.2 (11.6) 119.9 (7.4) 89,8 (9.1) 78.9 (8.9) 110.3 (5.6)

RI
Parboiled rice
LOQ 69.1 (6.7) 98.8 (12.3) 74.0 (5.6) 94.3 (7.1) 78.2 (16.7) 91.7 (9.6) 65.9 (10.8) 76.3 (9.8)

SC
5LOQ 97.6 (8.5) 82.4 (7.9) 107.9 (4.9) 116.3 (14.3) 60.7 (9.6) 70.8 (8.3) 61.7 (6.9) 67.1 (13.2)
10LOQ 83.2(14.2) 74.8 (9.3) 71.9 (10.9) 100.3 (15.2) 105.9 (88) 80.3 (11.3) 71.9 (17.4) 102.3 (4.9)
Polished rice
LOQ 62.6 (8.7) 84.4 (16.2) 100.1 (8.3)
U
62.8 (9.1) 89.2 (13.2) 67.3 (17.5) 91.4 (8.8) 65.3 (17.2)
AN
5LOQ 114.5 (11.8) 117.2 (8.9) 116.6 (7.1) 106.6 (6.8) 62.1 (18.9) 63.5 (9.6) 61.4 (7.2) 62.5 (11.6)
10LOQ 62.8 (6.1) 62.3 (5.1) 60.2 (17.3) 72.5 (12.8) 75.8 (6.7) 117.6 (8.7) 76,1 (5.2) 85.8 (13.3)
M

8 Results expressed as mean (RSD). n = 3 RSD relative standard deviation

D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
9 Table 4 – Aflatoxins reduction in rice after hydrothermal treatments
Thermal Aflatoxins reduction (%) Mean
Rice
source B1 B2 G1 G2 AFLAS
a a b a
Brown 88.9 (4.5) 80.4 (5.6) 67.4 (14.3) 88.3 (6.7) 81.3
Parboiled 85.8a (7.8) 90.4a (13.4) 90.5a (5.2) 79.9a (12.4) 86.7
Microwave
Polished 85.8a (9.3) 86.6a (5.7) 86.9ab (4.9) 82.7a (9,4) 85.5

PT
Brown 87.5a (11.2) 94.5a (8.3) 77.0ab (9.8) 76.1a (7.3) 83.8
Electric Parboiled 92.6a (6.4) 95.0a (3.1) 81.7ab (6.3) 93.0a (4.9) 90.6

RI
Plate
Polished 90.7a (8.1) 92.3a (6.4) 86.6ab (11.7) 78.8a (9.1) 87.1
10 Results presented as mean (n=3) and in wet basis. Different letters indicate significant

SC
11 difference (p<0.05) for factorial analysis of treatment*class.

U
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
12 Table 5 – Bioaccessibility of aflatoxins from cooked rice
Bioaccessibility (%)
Thermal
Mean
source Rice AFB1 AFB2 AFG1 AFG2
(AFLAS)
Brown 34.2c (4.5) 76.1a (8.3) 124.3a (5.1) 123.5a (4.8) 89.5
Parboiled 74.7b (7.8) 43.5b (15.6) 104.1bc (2.5) 56.4de (14.3) 69.7
Microwave

PT
Polished 66.8b (1.4) 63.9a (7.2) 72.0d (9.2) 76.2c (3.3) 69.7

Brown 16.4d (11.7) 15.0d (9.4) 32.8e (12.7) 71.4cd (7.1) 33.9

RI
Electric Parboiled 67.9b (6.1) 28.2c (11.9) 96.3c (6.8) 54.6e (11.2) 61.7
plate a cd ab b
Polished 89.7 (3.2) 26.1 (4.2) 118.4 (5.6) 106.0 (4.1) 58.5

SC
13 Results presented as mean (n=3). Different letters indicate significant difference (p<0.05) for
14 factorial analysis of treatment*class.
15

U
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT

MBrow

PT
RI
PlBrow

SC
Fig. 1

U
AN
M
D
TE
C EP
AC
 ACCEPTED MANUSCRIPT
Highlights

• Microwave oven and electric plate promoted a high level of aflatoxins reduction.

• Microwave treatment caused bioaccessibility (58%–100%) for aflatoxins B2, G1

and G2.

• The higher reduction of mycotoxin levels the lower its bioaccessibility.

PT
RI
U SC
AN
M
D
TE
C EP
AC