Effects of soaking process on arsenic and other mineral elements in brown rice

Journal Pre-proof Effects of soaking process on arsenic and other mineral elements in brown rice Fan Zhang, Fengying Gu, Huili Yan, Zhenyan He, Bolun Wang, Hao Liu, Tingting Yang, Feng Wang

PII:

S2213-4530(19)30233-2

DOI:

https://doi.org/10.1016/j.fshw.2020.01.005

Reference:

FSHW 212

To appear in:

Food Science and Human Wellness

Received Date:

15 November 2019

Revised Date:

27 December 2019

Accepted Date:

16 January 2020

Please cite this article as: Zhang F, Gu F, Yan H, He Z, Wang B, Liu H, Yang T, Wang F, Effects of soaking process on arsenic and other mineral elements in brown rice, Food Science and Human Wellness (2020), doi: https://doi.org/10.1016/j.fshw.2020.01.005

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Effects of soaking process on arsenic and other mineral elements in brown rice Fan Zhang

a, c

, Fengying Gu a, Huili Yan d, Zhenyan He d, Bolun Wang

a, c

, Hao Liu

a, c

, Tingting

Yang a, c, Feng Wanga, b * a Institute

Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing 100193, China

c Graduate

d Institute

school of Chinese Academy of Agricultural Sciences, Beijing 100081, China

of Botany, Chinese Academy of Sciences, Beijing 100093, China

*Corresponding

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b Key

of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Author: Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences /

Key Laboratory of Agro-Products Processing, Ministry of Agriculture, Beijing 100193, China.

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E-mail: [email protected] (Feng Wang)

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Telephone Number: 0086-010-62815647

Highlights: 1. The maximal reduction of arsenic was ~40%, inorganic arsenic accounting for ~80%. 2. Endosperm was a main part of arsenic removal in rice during soaking. 3. There was a consistent variation between elements in endosperm during soaking. 4. Soaking will be an alternative process to remove arsenic in brown rice.

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ABSTRACT

One of the main food crops in the world, rice can accumulate high levels of arsenic from flooded paddy soils, which seriously threatens human health. Soaking, a common processing

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method for brown rice products, especially for brown rice noodles, was investigated in this study.

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Japonica rice (Dao Hua Xiang No. 2) and Indica rice (Ye Xiang You No. 3) were selected for

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studying the effects of soaking on arsenic concentrations, species, and distributions. Results revealed that soaking can efficiently remove arsenic in these two rice varieties, and the main part

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of removal is endosperm with the maximal rate of ~40%. Inorganic arsenic (I-As) (~85%) is the main species of arsenic reduction. Meanwhile, the variations of four other elements (i.e., Mg, Ca,

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Zn, and Fe) were analyzed. Collectively, the findings of this study indicate that soaking can efficiently remove arsenic in brown rice under controlled soaking conditions, which thereby

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reduces the arsenic intake for brown rice customers. KEYWORDS: Brown rice; Soaking; Total arsenic; Arsenic species; Distribution

1. Introduction Brown rice is considered a whole grain cereal, as the outer layer is completely preserved. The bran layer contains many health-promoting components, including mineral elements, vitamins, and dietary fibers [1-6]. However, it is also a crop that easily accumulates arsenic from flooded paddy rice soils [7-9]. Due to industrial practices, massive waste has resulted in heavy arsenic

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pollution in the groundwater and soil of many countries, including China, and has specifically led to the accumulation of arsenic in brown rice [8, 10-12]. Furthermore, the climate change will also affect the rice yield and arsenic in paddy rice. The study indicated that the future climate

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conditions cause about twofold increase of I-As in grain [13]. Thus the toxic arsenic in brown rice

thereby enters the body through the food chain and results in a number of diseases [12, 14-17].

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The toxicity of arsenic depends on its chemical species [18, 19]. The toxicity of I-As ( As (III) and

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As (V) ) is higher than that of O-As ( DMA and MMA ) [20]. The International Agency for Research on Cancer has classified I-As as a group 1 carcinogen for humans [12, 21]. Thus many

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countries and organizations have limited the concentration of I-As in rice to ensure its security on the market, including the European Union, United Nations, World Health Organization, and China

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[22, 23]. Therefore, it is crucial to remove arsenic from rice, especially I-As in order to reduce

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toxicity.

To date, various methods, such as breeding low arsenic-accumulating rice cultivars, soil

remediation and water management, have been used to reduce the accumulation of arsenic in brown rice [24-26]. However, these methods have limited effectiveness and none of them can completely prevent the accumulation of arsenic into brown rice. There still is a vast number of high-arsenic brown rice in arsenic-contaminated areas worldwide every year. So various processes

are applied to reduce arsenic in brown rice. Many years ago, in South Asia and South China where the populace feeds on rice, natives would usually soak brown rice for the better quality [27, 28]. Indica rice starch has higher amylose than Japonica rice starch, which shows the higher melting and pasting viscosity but the higher gel hardness. So the Indica rice is usually used to make brown rice noodles after soaking, while Japonica rice is used to make cooked brown rice [29]. The conventional soaking process for cooking brown rice can achieve quick and uniform water

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absorption [3]. With advancements in technology, alternative methods have been applied for improving and simplifying the soaking process. In addition to improving its palatability, many studies have reported on improving nutritional value. For example, it was previously reported that

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soaking can significantly increase the GABA content of brown rice [30].

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However, only a few studies have focused on systematic soaking conditions and variations of

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arsenic concentrations, species, and distributions during the soaking process. A previous study demonstrated that soaking decreased arsenic by 18% without analyzing the arsenic species [31].

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Therefore, it is necessary to systematically focus on the effects of soaking on arsenic in brown rice. In this study, the effects of soaking on the variations of arsenic and other elements were evaluated

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in two different rice varieties. Different soaking conditions, including soaking temperature, time, mass ratio (water:rice), and acid percentage, were investigated. The findings of this study will

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undoubtedly serve as an important reference for arsenic removal in crops, including rice, and help domestic consumers to consume brown rice safely and healthily. 2. Materials and methods 2.1 Sample preparation In this study, two brown rice varieties were harvested in summer from two different

provinces of China at 2018. In order to verify the universality of methodology and representativeness of the results, Japonica rice (Dao Hua Xiang No. 2) from Harbin province, which is highly consumed, and Indica rice (Ye Xiang You No. 3) from Guangxi province, which has a wide planting area, were selected. Samples were mixed and washed three times with ultrapure water at a 2:1 ratio (water:rice g:g) to wash off surface dust, dried in an oven at 40℃ for 12 h, and sealed in valve bags at room temperature for further analysis.

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2.2 Soaked brown rice preparation

Fifteen grams of brown rice samples were mixed with soaking solutions in beakers under

different conditions. For the operability of soaking condition, mass ratio (3:1), temperature (30℃),

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and time (4 h) were chose as conventional conditions. Temperature experimental groups were

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soaked in ultrapure water at a 3:1 ratio (water:rice g:g) at different temperatures (i.e., 30℃, 40℃,

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50℃, 60℃, and 70℃) for 4 h. Time experimental groups were soaked in ultrapure water at a 3:1 ratio (water:rice g:g) at 30℃ for different lengths of time (i.e., 2, 4, 8, 24, and 48 h). Mass ratio

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experimental groups were soaked in ultrapure water at different ratios (i.e., 2:1, 3:1, 4:1, and 5:1 (water:rice g:g)) at 30℃ for 4 h. Acid percentage experimental groups were soaked in ultrapure

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water with different acid percentages (i.e., 0.5%, 1%, 1.5%, and 2%) at a 3:1 ratio (water:rice g:g) at 30℃ for 4 h. All groups were sealed during soaking. Two replicates of each group were

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analyzed for better precision. Temperature was controlled by water bath (HH-2, China). Glacial acetic acid (AR, China) was used to simulate an edible acidic environment. Sample were separated from the solution after soaking, washed with ultrapure water at a 3:1 ratio (water:rice g:g) and dried by vacuum freeze dryer (FD-1C-80, China). Element concentrations were calculated by dry weight. Then, samples were milled and homogenized by grinding machine (CK2000, China) at

1200 strokes/min for 90 s. Milled samples were stored in valve bags at room temperature for further analysis. 2.3 Analysis of element concentrations High performance liquid chromatography-atomic fluorescence spectrometry (ICP-MS) (Agilent 7700, USA) was used to detect the concentrations of As, Mg, Ca, Zn, and Fe after electric heating plate digestion. Milled samples were weighed (0.5 g) in a digestion tube, and 8 mL 70%

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HNO3 (BV-III, China) was added. Tubes were predigested at room temperature for 2 h and

digested at 110℃ for 3 h. Then 2 mL 30% H2O2 was added (BV-III, China) when the remaining digestion solution was at 2 mL. The tubes were removed and cooled to room temperature when

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0.5 mL remained. Lastly, ~50 mL ultrapure water was added to dilute the digestion solution; the

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dilution multiple was noted before injection. Instrumental settings and data acquisition parameters

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for the ICP–MS were as follows: (RF) power = 1550 W; carrier gas flow = 1.14 L/min; selected isotope = m/z 75. Each sample had three replicates. The certified reference rice flour (GBW (E)

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100348-100362, China) was used for method validation. 2.4 Analysis of arsenic species species

were

identified

and

quantified

by

high-performance

liquid

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Arsenic

chromatography-atomic fluorescence spectrometry (HPLC-AFS) (SA50-AFS8220, China) after

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extraction. One gram milled samples were weighed in a centrifuge tube mixed with 20 mL of 0.15 M HNO3 (BV-III, China) and extracted at 90℃ for 2.5 h. The supernatant was obtained by centrifugation at 6000 × g for 15 min and filtrated by 0.45 μm membrane filters. The HPLC equipment was connected to an anion-exchange column (4 × 250 mm). The mobile phase consisted of 15 mM (NH4)2HPO4 (pH = 6.0), and the flow rate was 1.0 mL/min. Then, 100 μL of

sample was injected. The conditions of AFS were as follows: voltage = 320 V; total lamp current = 90 mA; primary lamp current/assistant lamp current = 55/35; carrier solution = 20% hydrochloric acid (4 mL/min); carrier gas flow = 400 mL/min; and assistant gas flow = 400 mL/min. Each sample had three replicates. Standard solutions of arsenic species were bought from the National Institute of Metrology (China). The chromatogram and correlation coefficients of four arsenic standards solutions include arsenite (As (III)), dimethylarsine (DMA),

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monomethylated arsenic (MMA), and arsenate (As (V)), which are presented in Fig. 1.

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Fig. 1. Chromatogram and correlation coefficients of four arsenic standard solutions by HPLC-AFS. The peaks include As (III), DMA, MMA, and As (V) based on peak order. The

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standard curve correlation coefficients of each species are shown in the corresponding peaks. 2.5 In situ analysis

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Micro-X-ray fluorescence (μ-XRF) microspectroscopy was used to analyze the distribution of

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elements in the brown rice grains from the prior soaking experiment. The μ-XRF method was based on previous study [32, 33]. The μ-XRF experiment was performed at 4W1B Beamline, Beijing Synchrotron Radiation

Facility, which runs 2.5 GeV electrons with a current ranging from 150 to 250 mA. The incident X-ray energy was monochromatized by a W/B4C Double-Multilayer-Monochromator (DMM) at 15 keV and focused down to 50 μm in diameter by a polycapillary lens. The thick latitudinal

sections of brown rice grains were prepared and placed on 3M tape for the XRF analysis. Two-dimensional mapping was acquired by step-mode, where the sample was held on a precision motor-driven stage, scanning at 350 μm stepwise. The Si (Li) solid state detector was used to detect X-ray fluorescence emission lines with a live time of 15 s. The data reduction and process were performed by the PyMca 5.0.3 and Origin 8.0. 2.6 Quality assurance

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To avoid contamination, all reagents (i.e., soaking and digestion solutions, species extraction regents) and materials were determined by ICP-MS (Table 1). The element concentrations of the

external environment were quite low. Therefore, the external effects could be neglected. During

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experimentation, all vessels were soaked in 10% HNO3 for 48 h, rinsed three times with ultrapure

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water and dried before use.

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Table 1

Concentrations (mg/kg) of elements in materials and reagent blanks. Japonica rice

Reagent blank

(Dao Hua Xiang No. 2)

(Ye Xiang You No. 3)

0.28 ± 0.01

0.22 ± 0.01

< 0.00

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As

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Elements

Indica rice

1230.83 ± 28.69

1197.47 ± 28.71

< 0.13

Ca

124.13 ± 0.69

110.80 ± 1.62

< 1.44

Zn

19.80 ± 0.80

20.25 ± 0.14

< 0.24

Fe

11.67 ± 1.58

10.64 ± 1.44

< 0.49

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Mg

Values are the mean of three replicates; “± values” represent the standard errors; and “< mean values” represent the maximum concentrations of elements in reagents used during the

experiment. 2.7 Statistical analysis The presented data were obtained from repeated trials. Statistical analysis of the variance and correlation was performed by SAS software 9.4. Least significant difference tests were conducted for multiple comparisons at the p < 0.05 probability level. 3. Results and discussion

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3.1 Concentration variations of As, Mg, Ca, Zn, and Fe in brown rice during soaking

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Fig. 2. Concentration (mg/kg, dry weight) variations of As, Mg, Ca, Zn, and Fe in brown rice during soaking. A (A1, A2), B (B1, B2), C (C1, C2), D (D1, D2), and E (E1, E2) correspond with As, Mg, Ca, Zn, and Fe, respectively. Different colors represent different soaking conditions. Temperatures: 30℃, 40℃, 50℃, 60℃, and 70 ℃. Times: 0, 4, 8, 24, and 48 h. Mass ratios (water:rice g:g): 2:1, 3:1, 4:1, and 5:1. Acid percentage: 0%, 0.5%, 1.0%, 1.5%, and 2.0%. Values are presented as the mean of three replicates. Error bars represent the standard error.

The five elements exhibited different variation trends under different soaking conditions in Fig. 2. As was greatly affected by temperature and time, Mg and Ca were affected by time and acid percentage, Zn was affected by acid percentage, and Fe was unaffected. Particularly, Mg was affected by temperature in Japonica rice not in Indica rice. From the analysis of the diagrams, similar variation trends of different elements were observed under the same conditions, illustrating that some commonalities exist among the different elements, such as storage form, distribution, To obtain a better analysis of the five elements, significant differences

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and metabolic pathway.

of each condition were analyzed (Table 2). Table 2

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Significant difference analysis of the five elements in the two rice varieties under different soaking

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conditions.

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Japonica rice Mass P

Temperature

Time ratio (h)

< 0.0001***

Mg

0.0025*

Ca

0.5878

Fe

ratio

percentage

(g:g)

(%)

Time

Percentage

(℃)

Acid

(h)

(%)

0.2755

0.2674

< 0.0001***

< 0.0001***

0.7380

0.7388

< 0.0001***

0.2216

0.0166*

0.7618

< 0.0001***

0.0707

< 0.0001***

0.0134*

0.6309

0.0138*

0.2283

< 0.0001***

0.1952

0.0028*

0.1341

0.5569

0.7666

0.0003**

0.1546

0.1835

0.2138

0.0021*

0.0563

0.2353

0.2954

0.5925

0.1916

0.5701

0.4639

0.4960

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Zn

< 0.0001***

Temperature

Mass

(g:g)

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As

Acid

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(℃)

Indica rice

“*” p < 0.05; “**” p < 0.001; “***” p < 0.0001. Total As was significantly removed (p < 0.05) as soaking temperature increased. Notably, the

reduction of As in the two varieties was extremely obvious from 60℃ to 70℃, with a maximum reduction rate of 37.3% in Japonica rice and 32.9% in Indica rice at 70℃. Similar trends were observed for Mg in Japonica rice, but not Indica rice. Previous findings indicated that the pasting temperatures of Japonica and Indica rice flour were 66℃ and 79℃ respectively, as determined by differential scanning calorimeter (DSC Q200, USA) in Fig. 3. These results indicated that temperature affects the removal of As and Mg. This may because the heat involved in soaking can

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promote seed germination, destroy some enzymes and disrupt the structure of brown rice [4, 34].

Below the gelatinization temperature, the germination process was accelerated with the increase of temperature, which can result many channels to exchange substance. Some elements also can

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exchange with following these channels. When get to the gelatinization temperature, it may then

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increase the distance between starch chains and thereby produce more channels [35], which then

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form loose structures in brown rice and promote the dissolution of As and Mg.

Fig. 3. The pasting temperature of the two brown rice varieties was determined by a differential scanning calorimeter (DSC). Equilibrated at 30°C.

Ramp 10°C/min to 100 °C.

Total arsenic was significantly removed (p < 0.05) as time proceeded. Similar variation trends were observed for Mg and Ca in both rice varieties. The three element concentrations exhibited a downward trend during soaking. The maximum reduction rate of As was 18.3% at 48 h. From 0 to 4 h, the downward trends were similar to the water absorption characteristic curve of

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seed germination described in a previous study [36], which described a process of water

absorption from quick to slow and from less material exchange to more. The exchange of water can facilitate element dissolution. Moreover, its capacity was stable from 4 to 8 h. From 8 to 24 h

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or 48 h, element concentration continued to significantly decrease (p < 0.05). Meanwhile, an

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abnormal odor and turbidity were observed in the soaking solution, indicating that longtime

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soaking can cause germination and fermentation in brown rice. The process of soaking, germination and fermentation can increase the solubility of minerals in foods and soften grain

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texture [37, 38], as well as promote elements to decrease. These results indicate that the maximum reduction rate of As can compete with the polishing process without destroying the shape of

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brown rice.

As for the soaking mass ratio, no significant differences (p > 0.05) were detected among the

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five elements in both rice varieties. These results indicate that mass ratio is not a major factor. Thus, it is not necessary to remove As by increasing the mass ratio and keep other mineral elements intact by decreasing the mass ratio. By adding different percentages of acetic acid, significance differences of Mg, Ca, and Zn (p < 0.05) in both rice varieties were detected, but not for As or Fe (p > 0.05). These differing trends may be related to the storage form of elements.

Therefore, it is not recommended to remove As by adding acid, which results in the loss of other elements. According to the elements variations analysis, the variation results may be highly related to the process of seed germination [39]. During the water absorption, there are many channels in seed. These elements may

be decreased or permeate from solution following the channels.

Meanwhile, the internal and external concentration difference is also a restrictive factor. Different

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elements variation may be depend on their distribution, carriage conditions of channels and binding constant with some phytochelatins [40], which need a further verification. 3.2 Variations of As in brown rice during soaking

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I-As has higher toxicity than O-As. To better analyze the toxicity of brown rice, arsenic

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species were determined by HPLC-AFS. Comparing the two rice varieties, the variation trends of

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As were quite similar (Fig. 4). Analyses indicated that the downward trends of I-As were consistent with that of total As. The correlation coefficient reached 0.9, indicting high correlation

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between them. Analyses revealed that the soaking temperature and duration of time can remove As efficiently, especially I-As in Indica rice, indicating that soaking was an effective method for

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reducing I-As intake. However, O-As was contained at a stable content level during soaking in both rice varieties. The different variations indicate that I-As and O-As have the different storage

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form and metabolic mechanism. I-As may be chelate some large molecules in the form of ions, which can be dissociated easily with seed germination. While, O-As may be bind to the functional group of some proteins, which has a strong binding force. Therefore, O-As is difficult to remove during some conventional processes. Significant differences were detected between the properties of I-As and O-As, which can be applied to future studies of O-As.

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Fig. 4. Variations of As in brown rice during soaking (mg/kg, dry weight). (A, B) The effects of soaking temperature on As in the two varieties; (C, D) Soaking time. Pearson correlation

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coefficients are presented in the line charts. Values are presented as the mean of three replicates. Error bars represent the standard error. Brown rice, materials without any treatment; T-As, total arsenic; I-As, inorganic arsenic; and O-As, organic arsenic. 3.3 Distribution of five elements in brown rice grains Synchrotron radiation μ-XRF was employed to identify the distribution patterns of five

elements in brown rice grains without soaking and in grains soaked at 50℃ (Fig. 5). Arsenic was highly localized uniformly around the brown rice grain and embryo in both varieties, which is inconsistent with previous findings. These results illustrate that the specific distribution of As is influenced by the rice variety and plant region. Mg tended to be distributed evenly, Ca was mainly distributed in embryo with a little in endosperm, while Zn, and Fe were localized to the embryo of the rice grain. After soaking, the As and Mg concentrations significantly decreased in the

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endosperm, illustrating that the two elements may have similar storage forms and metabolic

channels. The variation of Ca was not quite significantly in graphic, while the data showed that the variation was significantly in 3.1. During soaking, the nutrients are metabolized into small

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molecules for plant growth with seed germination. Starch, the main carbohydrate in rice

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endosperm, is break down into short chain starch, which caused many small irregular voids [40].

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Therefore, following these voids, the As, Mg, and Ca may be decreased. This is good for human health because the As was removed efficiently without destroying the edible portion of endosperm.

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Meanwhile, there is a need to fortify Mg, and Ca in some specific products. For other elements, there were no significant decreases detected, which is consistent with the results described in 3.1.

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According to image analysis, the soaking process may be influence the elements distributed in endosperm easily. The main reason is that soaking process can promote the seed germination,

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which occurs in the endosperm mainly. Therefore, the elements distributed in endosperm have significant variations. With the seeds germinating and growing, the elements variation will be happen in embryo.

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Fig. 5. Distribution analyses of five elements in brown rice. A, Japonica rice; B, Indica rice. No,

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brown rice without any treatment; 50℃, brown rice soaked at 50℃ for 4 h. The depth of color represents the level of concentration. Red, high-concentration; Blue, low-concentration.

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4. Conclusions

By controlling the soaking conditions, total arsenic was efficiently removed from brown rice

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in this study. Briefly, the temperature and time significantly affected total arsenic and the

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maximum reduction rate was ~40%. The reduction of arsenic mainly involved I-As in the endosperm based on the analyses of species and distributions. Four other elements were also analyzed under the same conditions. Mg, and Ca have the similar trends with As, while Zn, and Fe were stable localized in embryo of the rice grain. According to the data analyses, the seed germination may be the main reason of elements variation.

Declaration of interest: none

Acknowledgments We would like to thank the Key laboratory of the Institute of Agro-Products Processing, Chinese Academy of Agricultural Sciences for experimental assistance. The μ-XRF beam time was performed by 4W1B Beamline of the Beijing Synchrotron Radiation Facility, Institute of

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High Energy Physics, Chinese Academy of Sciences. The staff members of 4W1B are acknowledged for their support in the measurements and reduction of the data. We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.

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This research was supported by the Special National Key Research and Development Plan [grant number 2016YFD0400204]; the Ministry of Science and Technology of the People's

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Republic of China [grant number 2015FY111300]; and the Technology Innovation Program of the

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Chinese Academy of Agricultural Sciences [grant number CAAS-ASTIP-201X-IAPPST]. References:

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