Original papers
Zinc Accumulation and Distribution in Germinated Brown Rice
2018 Volume 24 Issue 3 Pages 369-376
Details
2018 Volume 24 Issue 3 Pages 369-376
Zinc (Zn) is an essential trace element for human health. Organic zinc (O-Zn) is more security and has higher bioavailability than inorganic Zn. Brown rice with embryo and can germinate under the appropriate condition. During germination, brown rice could absorb exogenous inorganic Zn and transformed it into O-Zn by combining with macromolecules. In this study, germination conditions for zinc accumulation in germinated brown rice were investigated and the zinc distribution in various parts and macromolecule fractions were analyzed. Germination time, temperature and Zn2+ concentration significantly affected O-Zn content and ratio of O-Zn to total zinc (T-Zn) (O-Zn/T-Zn). The optimized condition was germinating for 3 d at 30.28°C after 200 mg/L of Zn2+ soaking. Under the optimum conditions, the maximum O-Zn content (304.71 µg/g) and O-Zn/T-Zn (86.21%) were obtained. Moreover, 21.19% of O-Zn was found in the protein fraction, 30.42% in the polysaccharide fraction and 1.24% in the nucleic acid fraction. T-Zn, O-Zn content and O-Zn/T-Zn declined substantially from the outer layers to the inner endosperm of Zn-enriched brown rice. O-Zn mainly combined with polysaccharide compared with protein and nucleic acid.
Zinc (Zn) is an essential trace element for human and animals and plays a key role in cell growth (Dang et al., 2010). Different from inorganic Zn, organic Zn (O-Zn) is characterized by stable chemical structure and better bioavailability which defined as the proportion of ingested Zn from food that can be absorbed and utilized for normal metabolic and physiological functions or storage (Plaimast et al., 2009). Zn deficiency can decrease immune function, learning ability, and inhibit mental development of children, even increase the risk of DNA injury and cancer development (Clemens, 2014; Prasad, 2017; Romualdo et al., 2016), thus more attention has been paid on the problem worldwide.
Rice is one of the most commonly consumed cereals, nearly 50% of the world population treat it as the staple food, and research on strengthening Zn nutrition in rice will sustainably alleviate Zn deficiency problem (Jeng et al., 2012). Studies have been conducted on improving Zn concentration in crops through breeding, transgenic approaches or the use of Zn-containing fertilizers (Boonchuay et al., 2013; Carvalho and Vasconcelos, 2013). However, it is not easily accepted by consumers because of high cost, long development cycle and potential soil pollution. Brown rice is the un-milled rice and has vitality (Patil and Khan, 2011). It could make some key enzymes activated and released under appropriate temperature and humidity condition. Steep brown rice using Zn solution firstly (Prom-U-thai et al., 2010), then germinate it in an incubator. During this procedure, brown rice absorbed exogenous inorganic Zn and transformed it into O-Zn by combining with macromolecules so as to increase O-Zn content and Zn bioavailability. It has been reported that obvious enrichment of mineral elements such as Zn (Yanyan et al., 2012), iron (Fe) (Wei et al., 2013) and selenium (Se) is possible in brown rice during germination using a Zn-containing, Fe-containing and Se-containing solution. Bioactive compounds enriched in brown rice were shown to vary greatly depending on the cultivar, soaking condition (Hualong et al., 2014) and germination condition (Fabiola et al., 2015). At present, it is available about germination condition for phytochemicals accumulation in brown rice (Caceres et al., 2014; Zhang et al., 2014). Some work has been done to understand Se accumulation in different protein fractions of Se-enriched brown rice and the molecular weight distribution of Se-containing proteins (Kunlun, 2010). However, it is unclear about the effect of germinated condition on Zn fortification and Zn binding capacity in different macromolecule fractions of brown rice.
The objectives of this study are to optimize germination condition to improve O-Zn content and the ratio of O-Zn to total zinc (T-Zn) (O-Zn/T-Zn), as well as investigate the binding-forms and spatial distribution of Zn in Zn-enriched brown rice obtained under the optimum condition.
Materials and reagents Brown rice named Wuyunjing 5055 was obtained from the Jiangsu Academy of Agricultural Science (JAAS) in China. The rice was harvested in 2015 in Jiangsu province of China. The brown rice was obtained by removal of the husk using a rice hulling machine (JGMJ8098, Shanghai Jiading Grain and Oil Instrument Co. Ltd., China) and stored in polyethylene bags at ȡ20°C until used.
Zn single element standard was obtained from National Center of Analysis and Testing for Nonferrous Metals and Electronic Materials; Zn sulfate heptahydrate was purchased from Sima-Aldrich Co. (St. Louis, USA).
Preparation of Zn-enriched brown rice Each brown rice sample (10 g) was sterilized with 1.0% (v/v) NaClO for 20 min and then rinsed three times with deionized water. Each sample was put into a 100 mL beaker and soaked at 25°C for 8 h with 50 mL of Zn2+ (ZnSO4) solution. The control was incubated with deionized water under the same condition. Then each sample was washed three times, and placed in a Petri dish evenly (15 cm in diameter) and covered with two layers of gauze. Each sample was germinated in darkness with different temperatures and 1 mL water was added every 12 h. Meanwhile, germinated seeds were collected after 0, 1, 2, 3 and 4 d germination. After being rinsed three times with deionized water, samples were oven-dried at 40°C and then stored at −20°C until analysis. Each treatment was repeated three times.
Single-factor experiment To optimize the appropriate condition for O-Zn accumulation in brown rice during germination, various germination times (1, 2, 3 and 4 days), germination temperatures (15, 20, 25, 30, 35 and 40°C) and Zn2+ concentrations (0, 50, 100, 150, 200 and 250 mg/L) were studied firstly. It has been detected that the concentration of Zn2+ from 0 to 250 mg/L did not affect the germination rate and sprout length of brown rice.
Optimization of germination condition for O-Zn accumulation Based on single factor experiment, the germination time (X1), germination temperature (X2) and Zn2+ concentration (X3) for O-Zn (Y1) and O-Zn/T-Zn (Y2) were optimized using response surface methodology (RSM). The factors and levels investigated in Box-Behnken design (BBD) are shown in Table 1. Seventeen combinations, including four replicates of the center points, were employed to evaluate the combined effects of variables on Y1 and Y2. The experimental results were analyzed by quadratic stepwise regression to fit the second-order equation:
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| Treatment | X1/Germination time(d) | X2/Germination temperature(°C) | X3/Zn2+ concentration(mg/L) | Y1/O-Zn content (µg/g DW) | Y2/O-Zn/T-Zn(%) | ||
|---|---|---|---|---|---|---|---|
| Real score | Predicted score | Real score | Predicted score | ||||
| 1 | 2 | 35 | 150 | 240.80 | 236.65 | 77.19 | 75.90 |
| 2 | 2 | 30 | 200 | 273.93 | 282.22 | 78.14 | 79.20 |
| 3 | 3 | 35 | 200 | 292.20 | 286.62 | 81.25 | 80.03 |
| 4 | 2 | 35 | 150 | 233.68 | 236.65 | 74.55 | 75.90 |
| 5 | 3 | 35 | 100 | 165.11 | 168.10 | 82.11 | 82.24 |
| 6 | 3 | 30 | 150 | 260.92 | 258.21 | 84.57 | 84.95 |
| 7 | 2 | 35 | 150 | 237.36 | 236.65 | 76.22 | 75.90 |
| 8 | 2 | 40 | 200 | 232.97 | 233.24 | 63.15 | 63.67 |
| 9 | 1 | 30 | 150 | 212.05 | 206.75 | 68.34 | 67.42 |
| 10 | 2 | 35 | 150 | 243.58 | 236.65 | 77.57 | 75.90 |
| 11 | 1 | 35 | 200 | 261.56 | 258.57 | 72.42 | 72.29 |
| 12 | 1 | 40 | 150 | 186.40 | 189.11 | 60.67 | 60.29 |
| 13 | 2 | 35 | 150 | 227.85 | 236.65 | 73.98 | 75.90 |
| 14 | 1 | 35 | 100 | 139.86 | 145.44 | 69.07 | 70.51 |
| 15 | 3 | 40 | 150 | 183.06 | 188.36 | 61.95 | 62.87 |
| 16 | 2 | 40 | 100 | 130.94 | 122.65 | 65.54 | 64.49 |
| 17 | 2 | 30 | 100 | 161.42 | 161.15 | 78.69 | 78.17 |
Where Y denotes the response observed for treatment combination X=(x1, x2, …, xp) for p factors, β0 represents the intercept, and the parameters of βi, βii, and βij represent the regression coefficients of variables for linear, quadratic, and interaction regression terms, respectively. An analysis of variance (ANOVA) table is generated to determine individual linear, quadratic, and interaction regression coefficients. The significance of polynomial relations was tested using Fisher's F test. The regression coefficients were used for statistical analyses to generate contour maps of the regression models.
Rice milling experiment Brown rice (100 g) was successively milled for 10, 20, 30, 40, 50 and 60 s using a laboratoryscale milling machine. Degree of milling (DOM) is defined as the percentage of the brown rice weight that is removed by the milling process. After each 10 s milling, the remaining rice and the abraded parts were collected carefully to determine DOM and Zn content, respectively. Fractions collected from 0–10 s, 10–20 s, 20–30 s, 30–40 s, 40–50 s, 50–60 s milling stages and the residue named I, II, III, IV, V, VI and WR (white rice), respectively.
Separation of O-Zn from Zn-enriched brown rice Zn-enriched brown rice was freeze-dried, ground and passed through a 0.15 mm pore-sized sieve. Rice flour (0.5 g) was dialyzed (molecular weight cut off of 100–500 Da) against deionized water for 96 h by changing the water every 12 h to remove Zn2+. After, Zn-containing compounds detected in the sample were considered as O-Zn (Zhao et al., 2004).
Separation of Zn-containing protein Each sample was freeze-dried, ground and passed through a 0.15 mm pore-sized sieve. The procedure of total protein extraction was according to Liu et al. (2011) with some modifications. Rice flour (3 g) was defatted with hexane, homogenized and extracted with 25 mL of NaOH (0.25 mol/L) at 25°C for 4 h under continuous stirring. The supernatant was obtained by centrifugation (10 000 g for 30 min) and the residue was extracted twice with 25 mL 0.25 mol/L of NaOH. Then the supernatant was combined and added ammonium sulphate (95% saturation) at 4°C. The precipitate was collected by centrifugation (6 000×g for 20 min) and then dissolved in deionized water. This solution was filtered (0.45 µm filter) and dialyzed (molecular weight cut off of 100–500 Da) against deionized water at 4°C three times to remove any other small molecules. Finally, the solution left was freeze-dried and stored at −20°C until used.
Separation of Zn-containing polysaccharide Each sample was freeze-dried, ground and passed through a 0.15 mm pore-sized sieve. Rice flour (3.0 g) was defatted with hexane, homogenized and extracted with 25 mL of NaOH (1.0 mol/L) at 60°C. After 4 h extraction, the supernatant was obtained by centrifugation (10 000×g for 30 min at 4°C) and the residue was washed and re-precipitated twice with 50 mL of NaOH (1.0 mol/L). Then the supernatant was combined and the protein in the supernatant was removed using the Sevag method (1:4, butanol and chloroform) (Zha et al., 2014). Ethanol was added to the supernatant to reach the concentration of 75% (v/v). After 12 h, the resultant precipitate was collected by centrifugation (6 000×g for 20 min) and then dissolved in 10.0 mL of deionized water. This solution was filtered and dialyzed against distilled water at 4°C three times to remove any other small molecules. The solution left was freeze-dried and stored at −20°C until used.
Separation of Zn-containing nucleic acid Each sample was freeze-dried, ground and passed through a 0.15 mm pore-sized sieve. The procedure of nucleic acid extraction was according to Zhao et al. (2004) with some modifications. Rice flour (3 g) was stirred into 100 mL of 12% NaCl at 95–100°C. After 2 h, the supernatant was obtained by filtration and the residue was extracted twice with 50 mL of 12% NaCl. Then the supernatant was combined and the protein in the supernatant was removed using the Sevag method. Finally, by adjusting the pH value of the supernatant to 2.5, the nucleic acids were precipitated at 4°C for 12 h. The precipitate was collected, freeze-dried, and stored at −20°C until used.
Determination of Zn content Contents of T-Zn, O-Zn, protein-bound Zn (Pr-Zn), polysaccharide-bound Zn (Ps-Zn) and nucleic acid-bound Zn (Nc-Zn) were determined by the methods described by Wei et al. (2012). Sample (1 g) was digested with 5 mL of a mixture of nitric acid and hydrogen peroxide (4:1, v/v) at 350°C for 2 h. After cooling, the digestion solution was transferred to a volumetric flask (100 mL), and the volume was filled with deionized water. The concentration of Zn in the sample was determined by inductively coupled plasma optical emission spectrometer (ICP-OES) (Optima 2100DV, Perkin-Elmer, Norwalk, CT).
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Statistical analysis The analysis of the variance was performed with the Statistical Analysis System software 8.2 (SAS, USA). Differences among means were evaluated using the Duncan's multiple range tests. Pearson's correlation coefficient values were established at significant levels of p < 0.05 and p < 0.01, respectively.
Effects of germination time, temperature and Zn2+ concentration on T-Zn, O-Zn contents and O-Zn/T-Zn Effects of germination time on T-Zn, O-Zn contents and O-Zn/T-Zn are shown in Fig. 1A. T-Zn content showed a decreasing trend with germination time; it may be that exogenous Zn2+ was absorbed during soaking process. As expected, O-Zn content and O-Zn/T-Zn significantly increased (p < 0.01) with germination time. This may be due to Zn accumulation at various germination stages through different metabolic pathways. O-Zn content had an obvious increase on the first and second day of germination, reached to 222.56 µg/g at 3 d and improved by 16-fold compared with raw brown rice (13.91 µg/g). Additionally, transformation speed of O-Zn was fast in the early period of germination which was differ from previous study (Kunlun, 2010). The reason probably was that some related enzymes activity were induced in soaking process which shorten germination time (Raes et al., 2014). The trend of O-Zn/T-Zn with germination time was similar to O-Zn content, and its maximize value was observed after 3 d germination, indicating that enzymes were activated during germination period to promote Zn2+ to combine with bioactive compounds in brown rice so as to form metal chelates.
Effect of germination time (A), germination temperature (B) and Zn2+ concentrations (C) on Zn accumulation and transformation of brown rice. (A): Zn-enriched germinated brown rice was obtained by germination with 250 mg/L Zn2+ concentration at 25°C. (B): Zn-enriched germinated brown rice was obtained by germination with 250 mg/L Zn2+ concentration for 3 d and (C): Zn-enriched germinated brown rice was obtained by germination at 35°C for 3 d. The results are expressed as mean ± SD with three replications. The different lowercase letters represent the significant difference at p < 0.05.
Effects of germination temperature on T-Zn, O-Zn contents and O-Zn/T-Zn are shown in Fig. 1B. Germination temperature had no significant (p > 0.05) effect on T-Zn contents with an exception (15°C), but had a significant (p < 0.01) impact on O-Zn content. At 35°C, O-Zn content and O-Zn/T-Zn in germinated brown rice reached the highest of 317.81 µg/g DW and 72.04%, respectively. The results suggested that under this condition some related enzymes were activated effectively. As a kind binding form of Zn, the Zn finger domain-containing proteins are the largest class of Zn-binding proteins in organisms, have been described as important regulators in plant responses to environmental stresses (lower temperature, etc.) (Huang et al., 2009), therefore indicates that high-temperature could accelerate Zn2+ to combine with biomacromolecule so as to improve O-Zn content in brown rice.
Fig. 1C shows the effects of Zn2+ concentration on T-Zn, O-Zn contents and O-Zn/T-Zn. A high correlation (r = 0.989) (p < 0.01) was observed between T-Zn content and Zn2+ concentration, and T-Zn content ranged from 19.33 to 420.10 µg/g DW germinating with 0–250 mg/L of ZnSO4. It was observed that the increase of Zn2+ concentration significantly (p < 0.01) improved T-Zn and O-Zn content in brown rice during germination, meanwhile gradually decreased O-Zn/T-Zn. This indicated that the increasing speed of O-Zn content was slower than T-Zn content and brown rice has limited bioaccumulation capacity for transforming the exogenous inorganic Zn to O-Zn. When Zn2+ concentration was 200 mg/L, O-Zn content reached the highest (328.1 µg/g DW), about 17-fold of the control (13.91 µg/g). High concentration of metal may cause toxicity in plant cells to inhibit plants development, even cause the death of plants by damaging membrane lipids, protein, pigments and nucleic acids (Weckx and Clijsters, 1997). However, it has been previously discussed that low concentration of metal slightly stimulated seed germination (Kranner and Colville, 2011; Prom-U-Thai et al., 2012). This could be resulted from an overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as nitric oxide (NO) in plants challenged by metals, leading to a slightly enhanced level of oxidative stress that stimulates germination (Kranner and Colville, 2011). In the present study, Zn2+ concentration achieved by preliminary experiment does not cause toxicity and has positive effect on seed germination.
Analysis experiment results of the response surface The optimal levels of the significant factors (germination time, germination temperature and Zn2+ concentration) and their interaction effects on O-Zn content and O-Zn/T-Zn were further explored by the BBD. By applying multiple regression analysis, the following two second-order polynomial equations were established to explain the O-Zn content (Eq. 3) and O-Zn/T-Zn (Eq. 4), respectively:
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The ANOVA for the BBD experiment gave relatively high F values (63.47 for O-Zn content and 34.99 for O-Zn/T-Zn), very low probability values (< 0.0001 for O-Zn content and O-Zn/T-Zn), and fairly large coefficients of determination (R2 = 0.9879 for O-Zn content and 0.9783 for O-Zn/T-Zn) as shown in Tables 2 and 3. A good agreement between experimental and predicted values implied that the mathematical model was very reliable for O-Zn content and O-Zn/T-Zn in the present study.
| Source | Sum of square | df | Mean square | F value | P valueprob > F | Significance |
|---|---|---|---|---|---|---|
| model | 35958.19 | 9 | 3995.35 | 63.47 | < 0.0001 | ** |
| X1 | 1285.75 | 1 | 1285.75 | 20.43 | 0.0027 | ** |
| X2 | 3825.94 | 1 | 3825.94 | 60.78 | 0.0001 | ** |
| X3 | 26834.34 | 1 | 26834.34 | 426.31 | < 0.0001 | ** |
| X1X2 | 681.47 | 1 | 681.47 | 10.83 | 0.0133 | * |
| X1X3 | 7.26 | 1 | 7.26 | 0.12 | 0.7441 | |
| X2X3 | 27.46 | 1 | 27.46 | 0.44 | 0.5301 | |
| X12 | 131.55 | 1 | 131.55 | 2.09 | 0.1915 | |
| X22 | 1762.06 | 1 | 1762.06 | 27.99 | 0.0011 | ** |
| X32 | 1129.98 | 1 | 1129.98 | 17.95 | 0.0039 | ** |
| residual | 440.62 | 7 | 62.95 | |||
| Lack of fit | 288.61 | 3 | 96.20 | 2.53 | 0.1956 | |
| Cor total | 36398.82 | 16 |
R2 = 0.9879, Adeq precision = 0.9723, coefficients of variation = 3.66%.
| Source | Sum of square | df | Mean square | F value | P valueprob > F | Significance |
|---|---|---|---|---|---|---|
| model | 842.34 | 9 | 93.59 | 39.22 | < 0.0001 | ** |
| X1 | 193.85 | 1 | 193.85 | 81.23 | < 0.0001 | |
| X2 | 426.76 | 1 | 426.76 | 178.84 | < 0.0001 | ** |
| X3 | 0.025 | 1 | 0.025 | 0.011 | 0.9209 | ** |
| X1X2 | 55.88 | 1 | 55.88 | 23.42 | 0.0019 | ** |
| X1X3 | 4.43 | 1 | 4.43 | 1.86 | 0.2152 | |
| X2X3 | 0.85 | 1 | 0.85 | 0.35 | 0.5702 | |
| X12 | 5.03 | 1 | 5.03 | 2.11 | 0.1897 | |
| X22 | 147.86 | 1 | 147.86 | 61.96 | 0.0001 | ** |
| X32 | 8.30 | 1 | 8.30 | 3.48 | 0.1044 | |
| Residual | 16.70 | 7 | 2.39 | |||
| Lack of fit | 6.64 | 3 | 2.21 | 0.88 | 0.5229 | |
| Cor total | 859.05 | 16 |
R2=0.9783, Adeq precision=0.9556, coefficients of variation=2.25%.
As shown in Tables 2 and 3, the linear effects of germination time, temperature and Zn2+ concentration were very significant (p < 0.01) for O-Zn content, and the linear effects of germination temperature and Zn2+ concentration were very significant (p < 0.01) for O-Zn/T-Zn (p < 0.01). Furthermore, germination time and temperature interacted significantly on O-Zn accumulation (p < 0.05), very significantly on O-Zn/T-Zn (p < 0.01). Because of the interaction effect of X1X2, the optimized condition of X2 was 30°C differ from single-factor experiment above (Fig. 1B).
The optimum condition and model verification According to the response surface method (RSM) test, the optimal incubation condition for T-Zn content and O-Zn/T-Zn was germinating at 30.28°C for 3 d after 200 mg/L of ZnSO4 soaking. Under this condition, the maximal O-Zn content and O-Zn/T-Zn detected were 304.71 µg/g and 86.15%, respectively. Verification of the model Eq. 3 and 4 were performed under the optimum condition for O-Zn content and O-Zn/T-Zn. The observed O-Zn content and O-Zn/T-Zn of Zn-enriched brown rice under the optimal condition was coincident with the predicted value in the model. The experimental statistics proved that the models were valid.
Distribution of Zn in Zn-enriched brown rice The Zn level in germinated brown rice with increase of DOM was presented in Fig. 2A. T-Zn, O-Zn contents and O-Zn/T-Zn in the abraded parts decreased significantly (p < 0.01) with milling time, suggesting that Zn content declined substantially from the outer layers to the inner endosperm. This may be due to that Zn2+ absorbed from external during soaking mainly distributed in outer layers, even though it was transported into inner endosperm slightly during germination. Zn distribution was un-uniform in Zn-enriched brown rice, which was similar to other minerals (Fe, Se, Mg, Ca, etc.) and anti-nutritional factors (phytic acid, etc.) distribution in raw brown rice (Itani et al., 2002). However, Zn distribution in raw brown rice was different from other minerals, it distributed throughout the kernel and with a higher concentration in the embryo (Liang et al., 2008).
Zn accumulation (A) in fractions of Zn-enriched brown rice and its loss during milling stages (B). After soaking with 200 mg/L of ZnSO4, brown rice was geminated at 30.3°C for 3 d. I, II, III, IV, V, VI and WR (white rice) refers to abraded parts collected from 0–10 s, 10–20 s, 20–30 s, 30–40 s, 40–50 s, 50–60 s at milling stages and the residue, respectively. BR refers to Zn-enriched brown rice. (B): Zn-enriched germinated brown rice was successively milled for 0, 10, 20, 30, 40, 50 and 60 s to obtain white rice with various DOMs. The different lowercase letters represent the significant difference at p < 0.05.
Zn-enriched brown rice was successively milled for 0, 10, 20, 30, 40, 50 and 60 s to obtain white rice with various DOMs. Relationship between DOM and loss of Zn was shown in Fig. 2B. Results showed that the loss of Zn increased significantly (p < 0.01) with an extension of milling time in Zn-enriched brown rice. It was especially remarkable at the early milling stages (0–40 s). This trend was in accordance with the DOM. It was clear that the DOM increased significantly (p < 0.01) with the milling time from 0 to 60 s. After being milled for 60 s, DOM reached to 17.86%. At the initial milling stages (0–40 s), the DOM increased sharply, but it become mitigatory when the milling time was longer than 40 s. This was similar with the previous report (Liu et al., 2009). The reason may be that the texture of the bran layer becomes harder gradually from outer to inner, whereas endosperm fraction was consistent in hardness.
Milling process removed a significant proportion of T-Zn and O-Zn from brown rice, resulting in a substantially reduction of T-Zn and O-Zn contents in the white rice. White rice is a normally consumed form in daily diet. After being milled for 60 s, an average of 55.25% and 49.95% in the loss of T-Zn and O-Zn was observed. Therefore, milling had an obvious and negative effect on Zn level of Zn-enriched brown rice.
Zn-binding forms in Zn-enriched brown rice On the basis of investigating distribution of Zn in brown rice, protein, polysaccharide and nucleic acid were extracted and their Zn accumulation was analyzed (Fig. 3). Pr-Zn concentration in the abraded parts decreased significantly (p < 0.01) with the milling time, suggesting that Pr-Zn level declined gradually from the surface to inner. Pr-Zn content was higher in outer bran layers than in endosperm, the distribution pattern was similar to that of protein in brown rice (Ning et al., 2010). However, Ps-Zn content increased at the early stage of milling (0–40 s), while had no significant change after milling for 40 s and reached the highest of 445.7 µg/g at 40–50 s. Ps-Zn distribution pattern was different from Pr-Zn, this may be due to that carbohydrates are mainly located in inner endosperm of brown rice. Furthermore, carbohydrates are important energy source for plant growth and its small organic metabolites are soluble in water (Chang et al., 2010), which accelerates combining speed of Zn2+ with polysaccharides during germination. Nc-Zn content of brown rice increased gradually with milling time and was the lowest compared with other binding-forms. This may due to the low level of nucleic acid in raw brown rice and adverse effect of the high temperature (90–95°C) on structural stability of Nc-Zn in the extraction process.
Different Zn-binding forms contents in fractions of Zn-enriched brown rice. After soaking with 200 mg/L of ZnSO4, brown rice was geminated at 30.3°C for 3 d. Pr-Zn, Ps-Zn, Nc-Zn refers to protein-bound Zn, polysaccharide-bound Zn, nucleic acid-bound Zn, respectively. I, II, III, IV, V, VI and WR (white rice) refers to abraded parts collected from 0–10 s, 10–20 s, 20–30 s, 30–40 s, 40–50 s, 50–60 s at milling stages and the residue, respectively. BR refers to Zn-enriched brown rice. The different lowercase letters represent the significant difference at p < 0.05.
The optimum condition for Zn accumulation in brown rice was germinating at 30.28°C for 3 d after 200 mg/L of ZnSO4 soaking. Zn content gradually decreased from outer bran to inner endosperm in Zn-enriched brown rice obtained under the optimal germination condition. In addition, Pr-Zn, Ps-Zn and Nc-Zn accounted 21.19%, 30.42% and 1.24% in O-Zn. Nearly a half of O-Zn remained in other components, which possibly included lipids and other low molecular weight compounds.
Acknowledgements This research was funded by Special Fund for Agro-scientific Research in the Public Interest (201313011-4).
