Determination of soluble sugar profile in rice

Accepted Manuscript Title: Determination of soluble sugar profile in rice Authors: Xianqiao Hu, Changyun Fang, Lin Lu, Zhanqiang Hu, Yafang Shao, Zhiwei Zhu PII: DOI: Reference:

S1570-0232(17)30074-0 http://dx.doi.org/doi:10.1016/j.jchromb.2017.05.001 CHROMB 20590

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

17-1-2017 28-4-2017 4-5-2017

Please cite this article as: Xianqiao Hu, Changyun Fang, Lin Lu, Zhanqiang Hu, Yafang Shao, Zhiwei Zhu, Determination of soluble sugar profile in rice, Journal of Chromatography Bhttp://dx.doi.org/10.1016/j.jchromb.2017.05.001 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.

Determination of soluble sugar profile in rice Xianqiao Hu1,2, Changyun Fang1,2, Lin Lu1,2, Zhanqiang Hu1,2, Yafang Shao1,2, Zhiwei Zhu1,2* 1

China National Rice Research Institute, Hangzhou 310006, China

2

Laboratory of Quality & Safety Risk Assessment for Rice (Hangzhou),

Ministry of Agriculture, Hangzhou 310006, China

*

Corresponding author. Tel.: +86 571 63370275; fax: +86 571 63370380. E-mail: [email protected].

Highlights  A method for analysis of soluble sugar profile in rice by using IC-PAD was presented.  Soluble glucose, fructose, sucrose, raffinose and maltose in rice were determined.  High performance was obtained for the method.

Abstract Soluble sugars in rice are the main components affecting sweetness taste of rice. In this paper, an accurate, precise and rapid method for simultaneous determination of multi soluble sugars in rice by using ion chromatography equipped with pulsed amperometric detector was 1

presented. Pretreatment and parameters of ion chromatography and pulsed amperometric detector were optimized. Regression coefficients (R) of 0.9998, 1.0000, 0.9979, 0.9998 and 0.9998 were obtained for glucose, fructose, sucrose, raffinose and maltose, respectively. The recovery ranges of five sugars were 92.9-112.0 % for milled rice matrix. Repeatability and reproducibility of the method were 0.8-9.7 % and 1.9-7.6 %, respectively. Method LODs of 3.1-34.6 μg∙g-1 were obtained for soluble sugars in milled rice matrix.

Keywords: soluble sugar, rice, ion chromatography, pulsed amperometric detector

1. Introduction Rice is one of the most important staple foods for world population[1]. The taste of rice is primarily associated with soluble components such as soluble sugars and amino acids. It was reported that soluble sugars in rice such as glucose and sucrose are the main components affecting sweetness taste of rice[2,3]. Hence, soluble sugar content and profile is an important index to rice quality especially taste quality. Sucrose, glucose, fructose, maltose and raffinose are the major soluble sugars in rice[4-6]. It’s necessary to assess the soluble sugar profile of rice kernels for rice quality assessment. 2

There are many methods reported to determine sugar content in food[7,8]. Hydrometer[9], refractometer[10] were used to measure the total sugar content. Electronic tongue[2,11] and near infrared spectroscopy (NIR)[12] were also applied for assessing the sweetness of samples. However, the results were strongly influenced by the prediction training. High pressure liquid chromatography(HPLC)[9,13], gas chromatography (GC)[14] and capillary electrophoresis were also used to determine sugar content in samples. As sugars do not absorb ultraviolet or visible wavelengths, it is not possible to determine sugars using HPLC with ultraviolet-visible detector unless proper derivatization was implemented. Evaporative light-scattering detector (ELSD) and differential refractive index detector (RID) are universal detectors, hence they were widely used for sugar analysis, needing no derivatization.[9-12] ELSD is based on the ability of particles to cause photon scattering, and hence, it can detect most compounds less volatile than mobile phase[9]. However, high purity quality of mobile solution was required for stable baseline. ELSD calibration response curve is non-linear which might cause errors in quantification[7]. Furthermore, it possesses relatively low sensitivity, and consequently is not suitable for trace analysis. RID works as a differential refractometer that measures the difference in refraction index of eluent induced by solute[15]. The signal is highly dependent on wavelength, temperature and density of solute, hence gradient elution is not applicable 3

to RID. Pulsed amperometric detector (PAD) is also used for detecting sugars and fructan in fruit and vegetable extracts. Compared to ELSD and RID, PAD is seen to more sensitive and reliable as a detector for carbohydrates. Besides, PAD is not sensitive to the changes of mobile solution. Hence, PAD is a more suitable detector for sugar analysis. As aqueous solution is much more appropriate for electron conductivity during redox at the electrodes than organic solution, PAD is usually coupled with ion chromatography (IC)

[16-18]

. IC-PAD method has been

applied for sugar analysis in inulin[17,18], olive plant[19], rice wine[20], raw sugar[21]. However, it has not been applied for sugar analysis in rice. Soluble sugars in rice were extracted and concentrated before introduced into HPLC with RID[2,6], making the determination tedious. This work reported a sensitive and reliable method for determining multi soluble sugars in rice by using IC-PAD. The pretreatment for rice sample and the condition for separation and detection were optimized. The performance of the IC-PAD method was investigated.

2. Material and Methods 2.1 Chemicals and materials High-purity (≥99%) glucose, fructose and sucrose were obtained from National Institute of metrology, China. High-purity (≥99%) maltose and raffinose were purchased from Dr. Ehrenstorfer GmbH, Germany. 30 % 4

sodium hydroxide aqueous (NaOH) were purchased from Merck KGaA, Germany. All solutions were prepared with deionized water using a Millipore advantage 10 system (Millipore Co. USA). 2.2 Rice samples and pretreatment Five rice samples were provided by Rice Product Quality Supervision and Inspection Center, Ministry of Agriculture. Before testing, rice samples were husked. Then half of husked rice sample were milled until most of bran and part of embryo had been removed. After that, both husked rice samples and milled rice samples were ground into flour by using a Cyclotec 1093 sample mill(Foss Tecator, Sweden). The rice flour samples were stored in -20 ºC before use. During extraction, an aliquot of flour sample (0.25 g for husked rice flour while 0.5 g for milled rice flour) was weighed and extracted with 10 mL of 50 % ethanol aqueous solution. The mixture was kept on a mechanical shaker for 30 min at the room temperature. After centrifuged at 3000 rpm for 10 min, the supernatant was collected. The process was repeated twice and supernatants collected were evenly mixed. Finally, the mixed supernatant was filtered through a 0.45 μm filter and ready for the following measurement. 2.3 Determination of soluble sugars Soluble sugars were determined by 850 Professional IC equipped with 871 Advanced Bioscan (Metrohm, Shanghai, China). IC separation 5

was performed on a Metrosep Carb 1 column (5.0 μm，150 mm × 4.0 mm) with a Metrosep guard column. The isocratic mobile phase was consisted of solvent A (250 mmol·L-1 NaOH) and solvent B (H2O) at a ratio of 40:60. The flow rate for mobile phase solution was 1 mL·min-1. The eluted analytes were detected and quantified by an 871 Advanced Bioscan with PAD mode (Metrohm, Shanghai, China). Gold electrode and Ag/AgCl electrode were used as working electrode and reference electrode, respectively. The detail parameters for PAD was as following: E1=0.1 V，t1=0.4 s，E2=0.7 V，t2=0.2 s，E3=-0.1 V，t3=0.4 s，tsample=40 ms. Here, E1, E2, and E3 were defined as the detection potential, oxidation cleaning potential and reduction cleaning potential, respectively, and t1, t2 and t3 represented time duration to apply E1, E2 and E3, respectively. Tsample was defined as detection time at the end of E1.

3. Results and Discussion 3.1 Sample pretreatment Three extraction methods were compared by repeating extraction for twice. With shake treatment method, the mixture was kept on a mechanical shaker for 30 min. The mixture was treated with ultrasonic for 30 min in ultrasonic treatment method. However, it was found that sample flour was deposited at the bottom of centrifuge tube due to gravity during ultrasonic treatment. Hence, in ultrasonic and shake treatment 6

method, the mixture was kept in an ultrasonic instrument for 30 min during which the mixture was shook with a vortex shaker for 5 times to avoid sample flour being deposited at the bottom. The results were showed in Fig.1a. It was found that ultrasonic treatment method resulted in the lowest sugar content. It was mainly because of incomplete extraction caused by the deposition of sample flour at the bottom. Shake treatment method resulted in highest contents of all sugars except sucrose content. The highest sucrose content was obtained by using ultrasonic and shake treatment method. However, the operation of ultrasonic and shake treatment method is quite cumbersome. Hence, shake treatment method was chosen taking account of extraction efficiency and ease of operation. The number of extraction times was also investigated. With extraction times increased from 1 to 2, glucose, fructose, sucrose and maltose content increased while raffinose content decreased (Fig.1b). However, the increase flatted when extraction times increased from 2 to 4. Hence, the extraction was repeated twice in the following work. 3.2 Detection potential Detection potential had great effect on the signals of object sugars (Fig.2a). The peak areas of glucose and fructose increased rapidly with the detection potential increased from -0.05 V to 0.1 V, and then leveled off. Similar trends were observed for other three sugars, except for the turning points of 0.2 V for sucrose and raffinose, and 0.05 V for maltose. 7

Meanwhile, detection potential affect the baseline and baseline noise as well. As seen from Fig.2b, sharp decline of baseline drift in 30 min was observed with detection potential increased from -0.05 to 0.05 V, and then it leveled off at the detection potential of 0.05 to 0.20V, and finally the baseline drift increased with the increase of detection potential up to test voltage. A lowest baseline drift was obtained at the detection potential of 0.1 V. The baseline noise sharply decreased with the increase of detection potential up to 0.1 V, and then sharply increased again with the increased of detection potential up to test voltage. A lowest of baseline noise was obtained at detection potential of 0.1 V. Hence, a detection potential of 0.1 V was employed to balance signal sensitivities of all sugars and baseline condition. 3.3 Mobile phase solution A solution containing five sugars was applied to investigate the retention behaviors. The mobile phase was consisted of solvent A (250 mmol·L-1 NaOH) and solvent B (H2O). The proportion of solvent A ranged from 10 % to 60 %. Hence, the concentration of NaOH in mobile phase solution was from 25-150 mmol·L-1. The retention time of five sugars were showed in Fig.S1. There might be some difficulty in separations among fructose, sucrose and raffinose. Hence, the retention behaviors of fructose, sucrose and raffinose were investigated as representative compounds. As showed in Fig.3, the resolution of sucrose 8

and raffinose decreased with the increase of NaOH concentration. However, opposite trend was observed with the resolution of fructose and sucrose. A NaOH concentration of 100 mmol·L-1 (40 % solvent A) was chosen due to the most time-saving condition with a resolution higher than 1.5. 3.4 Analytical performance of developed method A series of standard solutions containing glucose, fructose, sucrose, raffinose and maltose (Table S1) were selected to investigate the analytical performance of developed method according to each soluble sugar content in rice. As seen from Table 1, regression coefficients (R) of 0.9998, 1.0000, 0.9979, 0.9998 and 0.9998 were obtained for glucose, fructose, sucrose, raffinose and maltose, respectively. Method limits of determination (LOD) of 18.8, 6.3, 34.6, 3.1 and 3.1 μg∙g-1 were obtained for glucose, fructose, sucrose, raffinose and maltose, respectively, by determining one milled rice sample for 7 repeats. Meanwhile, method LODs of sugars in husked rice matrix were 28.3, 3.1, 141.4, 22.0 and 3.1 μg∙g-1. The accuracy of developed method was evaluated by recovery assay in both milled rice matrix and husked rice matrix. Milled rice samples and husked rice samples were spiked with standard solutions containing five sugars at low, intermediate and high concentration levels, respectively, and then determined with developed method for six repeats. 9

The recovery ranges of five sugars were 92.9-112.0 % for milled rice matrix and 90.7-107.9 % for husked rice matrix (Table 1), indicating the accuracy of developed method . The precision of developed method was also evaluated. As seen from Table 2, the precision of developed method was very good in determining sugar content in rice. Repeatability (n=6) for milled rice samples were 0.8-3.0 %, 2.9-7.5 %, 1.0-3.3 %, 1.8-4.7 % and 2.9-8.6 %, while reproducibility for three different persons were 2.2-5.7 %, 2.8-5.3 %, 1.9-3.3 %, 2.8-3.2 % and 4.7-7.5 % for glucose, fructose, sucrose, raffinose and maltose, respectively. Repeatability (n=6) for husked rice were 2.8-8.8 %, 2.1-6.3 %, 1.0-7.1 %, 2.0-9.7 % and 2.4-8.9 %, while reproducibility (n=3) were 3.2-5.9 %, 3.5-6.7 %, 1.7-4.1 %, 2.0-5.9 % and 3.0-7.6 % for glucose, fructose, sucrose, raffinose and maltose, respectively. Both repeatability and reproducibility were all lower than 10 %, indicating the acceptable precision of the method for soluble sugar content analysis.

4. Conclusion Soluble sugars are the main components for sweetness taste of rice. In this paper, a sensitive and reliable method for simultaneous determination of soluble sugars in rice by using IC-PAD was presented. The method was accurate, precise and rapid. The major soluble sugars 10

including glucose, fructose, sucrose, raffinose and maltose were determined in both husked and milled rice samples. Regression coefficients of 0.9979-1.0000 were obtained for five sugars. The recovery ranges of five sugars were 92.9-112.0 % for milled rice matrix and 90.7-107.9 % for husked rice matrix. Repeatability and reproducibility of the method were 0.8-9.7 % and 1.9-7.6 %, respectively. Method LODs of 18.8, 6.3, 34.6, 3.1 and 3.1 μg∙g-1 were obtained for glucose, fructose, sucrose, raffinose and maltose in milled rice matrix, and 28.3, 3.1, 141.4, 22.0 and 3.1 μg∙g-1 in husked rice matrix. The developed method was applicable to sugar analysis for rice, and useful for sweetness analysis for rice quality in future.

Acknowledgment This work was funded by Zhejiang Provincial Natural Science Foundation of China (grant No. LQ15C200007), Special Fund of Chinese Central Government for Basic Scientific Research Operations in Commonweal Research Institutes (project No. 2014RG006-4 and 2014RG006-1), and Science and Technology Planning Project of Zhejiang Province, China ( project No. 2016C37039), National Natural Science Foundation of China (project No. 31500246), and the National Key Research and Development Program of China(2016YFF0201803).

11

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Figure Captions

Figure 1 Influence of pretreatment on the final result. (a) sugar contents obtained with three sample extraction methods; (b) sugar contents obtained with different times.

Figure 2 Effect of detection potential on signals of five sugars and baseline condition. (a) effect of detection potential on signals of five sugars; (b) effect of detection potential on baseline drift and baseline noise.

Figure 3 Effect of NaOH concentration in mobile phase solution on the resolutions.

15

Figure 1

(a)

(b) 16

Figure 2

(a)

(b) 17

Figure 3

18

Table 1 Linear equation, correlation coefficient, method LOD and recovery of developed method Linear equation

Linear range(mg∙L-1)

R2

Method LOD1 (μg∙g-1)

Method LOD2 (μg∙g-1)

Recovery1 (%)

Recovery2 (%)

Glucose

y=6.9000x+1.9595

1-50

0.9996

18.8

28.3

106.6-112.0

103.6-107.9

Fructose

y=4.4973x+0.0181

0.2-10

1.0000

6.3

3.1

92.9-99.4

90.7-102.3

Sucrose

y=4.1143x+8.3690

2-100

0.9959

34.6

141.4

95.4-99.4

93.8-101.5

Raffinose

y=4.0642x+1.3252

0.5-25

0.9996

3.1

22.0

10.7-111.7

97.5-103.7

Maltose

y=4.4988x+0.2098

0.5-25

0.9997

3.1

3.1

96.4-99.1

92.2-106.2

1

: obtained with milled rice matrix;

2

: obtained with husked rice matrix.

19

Table 2 Repeatability and reproducibility of developed method Milled rice samples

glucose

fructose

sucrose

raffinose

maltose

Husked rice samples

Sample

Mean

Repeatability

Reproducibility

Mean

Repeatability

Reproducibility

no.

(mg∙g-1)

(%, n=6)

(%, n=3)

(mg∙g-1)

(%, n=6)

(%, n=3)

1

0.220

3.0

3.3

0.555

2.8

3.5

2

0.399

1.4

5.7

0.604

6.7

4.9

3

0.240

2.8

2.9

0.631

6.7

5.4

4

0.646

2.5

2.2

1.707

8.8

5.9

5

2.286

0.8

2.6

3.818

3.2

3.2

1

0.028

7.5

5.3

0.049

3.6

3.5

2

0.050

2.9

2.8

0.066

4.3

6.7

3

0.046

3.4

4.0

0.083

5.2

5.1

4

0.029

4.5

4.3

0.045

6.3

6.2

5

0.114

3.6

3.7

0.116

2.1

4.0

1

1.462

1.4

3.3

5.864

1.8

2.2

2

1.740

1.3

2.7

5.238

2.2

1.8

3

1.382

1.3

3.3

6.105

1.8

1.7

4

2.063

3.3

3.3

6.301

7.1

4.1

5

5.524

1.0

1.9

10.301

1.0

2.5

1

0.149

1.8

3.0

0.807

3.2

3.3

2

0.192

3.2

2.9

0.774

2.0

2.0

3

0.095

2.8

2.8

0.568

2.4

2.0

4

0.135

4.7

2.9

0.586

9.7

5.9

5

0.251

2.6

3.2

0.630

2.9

4.8

1

0.029

5.4

7.1

0.178

6.2

6.4

2

0.068

2.9

6.8

0.321

5.1

6.2

3

0.040

5.1

4.7

0.235

8.9

7.6

4

0.113

8.6

7.5

1.220

3.8

5.3

20

5

1.335

4.2

5.4

21

3.924

2.4

3.0