Isolated rice starch fine structures and pasting properties changes during pre-germination of three Thai paddy (Oryza sativa L.) cultivars

Journal of Cereal Science 70 (2016) 116e122

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Isolated rice starch fine structures and pasting properties changes during pre-germination of three Thai paddy (Oryza sativa L.) cultivars Hathairat Pinkaew a, Masubon Thongngam a, Ya-Jane Wang b, Onanong Naivikul a, * a

Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, 50 Ngam Wong Wan Rd., Lat Yao, Chatuchak, Bangkok 10900, Thailand b Department of Food Science, 2650 N Young Ave., Office: N-214, University of Arkansas, Fayetteville, AR, 72704, USA

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 September 2015 Received in revised form 19 April 2016 Accepted 6 May 2016 Available online 7 May 2016

This research aimed to investigate fine structural changes and pasting properties of isolated rice starches during pre-germination. Three stages of pre-germinated brown rice (PGBR) and flour were prepared from three different Thai paddy cultivars. Changes in a-amylase activities of flour were determined. Starch was isolated from the flour by alkali extraction. The molecular size distributions, amylopectin branch chain length distributions, and pasting properties of three-stage PGBR starches were compared to ungerminated brown rice (UGBR) starches. During pre-germination, the a-amylase activities of PGBR flour from all rice cultivars were increased. Isolated UGBR starches from the three rice cultivars had higher starch yields than three-stage PGBR starches. HPSEC-RI results showed different molecular size distributions; the amylose contents of KDML105 and RD31were reduced from 15.5 to 12.7% and 18.6 to 15.8% at the 1st and 2nd stages of pre-germination, respectively. HPAEC-PAD results indicated that isolated three-stage PGBR starches from three rice cultivars had lower short A chains amylopectin branch chains than their UGBR starches. Pre-germination affected pasting properties in which the peak viscosities changed at the 1st stage of pre-germination for RD6 (3662 cP) and the 2nd stage of pregermination for KDML105 (3860 cP) and RD31 (2279 cP) cultivars. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Pre-germinated brown rice starches Starch molecular size distributions Amylopectin branch chain length distributions Pasting properties

1. Introduction Pre-germinated brown rice (PGBR) could be produced from both paddy and brown rice by soaking paddy in water at 30
C for 12e14 h so that the moisture content of the soaked paddy was at least 30%. The soaked paddy was incubated at the same temperature and at 85% relative humidity (RH) until three stages of embryonic growth length (EGL) were achieved: first stage (0.5e1 mm, 60e70% of pre-germination), second stage (1e2 mm, 71e80% of pre-germination), and third stage (2e3 mm, more than 80% of pregermination) (Panchan and Naivikul, 2009). Researchers found that

Abbreviations: AAC, apparent amylose content; CL, average chain length; DP, degree of polymerization; EGL, embryonic growth length; GBR, germinated brown rice; HPAEC-PAD, high performance anion-exchange chromatography equipped with a pulsed amperometric detector; HPSEC-RI, high performance size-exclusion chromatography with a refractive index detector; KDML105, Khao Dawk Mali105; PGBR, pre-germinated brown rice; RD31, Rice Division31; RD6, Rice Division6; RH, relative humidity; SN, stirring number; UGBR, ungerminated brown rice. * Corresponding author. E-mail address: [email protected] (O. Naivikul). http://dx.doi.org/10.1016/j.jcs.2016.05.009 0733-5210/© 2016 Elsevier Ltd. All rights reserved.

the suitable soaking time for Thai Indica paddy (Suphan Buri1, 25.12% amylose content) was 14 h and the total pre-germination times to achieve three stages of EGL at first, second, and third stage were 28, 32, and 38 h, respectively. During rice germination, the dormant hydrolytic enzymes especially a-amylase was activated. Afterwards, endosperm starch is gradually hydrolyzed by aamylase to supply energy for germination (Kaneko et al., 2002). Germination has been widely reported to affect many chemical constituents of brown rice starches. In comparison to ungerminated brown rice (UGBR) starch (23.83% amylose content), Musa et al. (2011) reported that germinated Malaysian brown Indica rice starch (mixed varieties of MR219 and MR220) showed lower amylose content (21.78%). Researchers suggested that the reduction of amylose likely resulted from a-amylase. The rice cultivars and germination conditions are the two main factors affecting the reduction of amylose contents in germinated brown rice (GBR) starches. Xu et al. (2012) evaluated the change in starch yield after long-grain brown rice (17.39% amylose content) was germinated. They found that the isolated GBR starch yield was reduced from 46.75 to 42.12% on the grain mass basis after long-grain brown rice was soaked in distilled water at room temperature for 12 h and

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germinated at 30
C for 24 h with 65% RH in a biological oxygen demand (BOD) incubator. Wu et al. (2013) reported the changes in the amylopectin/amylose ratios of GBR starches from Chinese brown rice (long-grain Indica, medium-grain Japonica, and medium-grain waxy) cultivars. The amylopectin/amylose ratios of GBR starches from long-grain Indica (2.57e2.73) and medium-grain Japonica (4.13e4.69) brown rice increased, while the amylopectin/ amylose ratio of GBR starch from the medium-grain waxy (18.26e18.28) brown rice remained constant after five days of germination. Germination affected the amylopectin branch chain length of germinated long-grain brown rice. The proportion of short-chain length amylopectin decreased while the proportions of medium and long-chain length amylopectin slightly increased (Xu et al., 2012). In addition, germination induced remarkably changes in peak viscosities of GBR starches. Increased peak viscosity of GBR starch was reported by Wu et al. (2013) who suggested that an increase in peak viscosity was negatively correlated with the reduction of amylose content during germination. However, decreased peak viscosity of GBR starch was reported by Xu et al. (2012). The differences in peak viscosities of GBR starches may be due to the effects of the rice cultivars and the germination conditions in the experiments. Previously, researchers had paid attention on properties of GBR. However, information regarding to effects of germination on selected properties of germinated paddy is limited. In addition, since amylose and amylopectin contents and structures influence rice properties, it is crucial to determine the changes of these components during early stage of germination of different rice cultivars. The objective of this research was to investigate the incubation times for pre-germination of three Thai paddy cultivars with different amylose contents. The chemical compositions, fine structures, and pasting properties of the three-stage PGBR starches were examined in comparison to UGBR starches, to provide useful knowledge about the impact of the pre-germination process for starch bio-modification. 2. Materials and methods 2.1. Materials Three Thai paddy cultivars (Oryza sativa L.): Rice Division6 (RD6, waxy); Khao Dawk Mali105 (KDML105, low amylose); and Rice Division31 (RD31, high amylose) rice from the 2011 crop were obtained from the Rice Research Center (Bangkok, Thailand). Isoamylase (specific activity ¼ 210 units/mg protein) and pullulanase (specific activity ¼ 37 units/mg protein) were purchased from Megazyme International Ireland Ltd. (Wicklow, Ireland). Analyticalgrade chemicals were used in this research unless otherwise noted. 2.2. Pre-germinated paddy and flour preparation Paddy was soaked in 1.7% sodium chloride for 30 min to suppressed mold growth, followed by washing 4 times in tap water. The pre-germination process was conducted by soaking the paddy grains in tap water at a controlled temperature of 30
C for 12 h. The soaking water was changed every 6 h in order to prevent microorganism fermentation. The moisture content of soaked paddy was measured by air-oven methods according to Approved Method 4415A (AACC, 2000). Soaked paddy was removed and allowed to incubate at a controlled temperature of 30
C and 85% RH until the three stages of EGL were achieved: first (0.5e1 mm, 60e70% of pregermination), second (1e2 mm, 71e80% of pre-germination) and third (2e3 mm, more than 80% of pre-germination) stages. After that, each pre-germinated paddy was dried at 45 ± 10
C until the moisture content was less than 12% (Pinkaew and Naivikul, 2012).

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Ungerminated and three stages of pre-germinated paddy were dehulled prior to be finely ground using a pin mill (Alpine Ausburg 160 Z 1979, Germany). The flour from UGBR and the three stages of PGBR were passed through a 100-mesh sieve, packed in plastic bags, and stored at 18
C before further use. 2.3. Rice starch isolation Flour (200 g each) was defatted with 800 mL hexane and dried in a fume hood at room temperature for at least 24 h (Agboola et al., 2005). The defatted flour was then used for starch isolation by alkali extraction method (Suksomboon and Naivikul, 2006). The defatted flour (200 g each) was mixed with 0.2% (0.05 M) NaOH (500 mL) at 25
C for 3 h. The slurry was filtered through a 200-mesh laboratory test sieve (75 mm opening) in order to remove some large particles. The filtrate was centrifuged at 3000g for 20 min, the supernatant was discarded, and the sediment was washed twice with distilled water (500 mL) and centrifuged. The residue was suspended in distilled water and adjusted to pH 7 by adding 1 M HCl, and the slurry was then centrifuged. The supernatant was discarded, and the yellow tailings layer over the starch layer (white layer) was carefully scraped away and discarded. The starch was washed three times with distilled water (50 mL), and then dried overnight at 40
C in a tray dryer. The dried starch was finely ground using a hammer mill grinder with a 0.5-mm sieve. The starch samples were then passed through a 100-mesh sieve. The weight of this isolated rice starch was determined, and the percentage of starch yield was calculated based on the difference between the weights of UGBR and of the three stages of PGBR flour (dry basis, db). The isolated rice starch was packed in plastic bags, and stored at 18
C before further analyses. 2.4. Experimental design A 3  4 full factorial in completely randomized design (CRD) was used as the experimental design in this research. The main effects were three rice cultivars (RD6, KDML105, and RD31) and four pregermination durations (ungerminated, pre-germinated at the first, second, and third stage). Each treatment combination had three replicates. 2.5. Chemical composition The moisture content was measured by air-oven methods according to Approved Method 44-15A (AACC, 2000). Crude protein was measured by a Kjeldahl method according to Approved Method 46-11A (AACC, 2000) using the conversion factor of 5.95 to convert nitrogen content to crude protein. Crude fat was measured according to Approved Method 30-20 (AACC, 2000) using a Soxtec apparatus and hexane as the solvent. The apparent amylose content (AAC) was measured using the colorimetric method of Juliano (1971). All chemical compositions were measured in duplicate. 2.6. a-amylase activity measurement

a-amylase activity was measured in flour using a Rapid Visco Analyzer (RVA; Newport Scientific Pty, Ltd., Australia) following the Approved Method 22-08 (AACC, 2000). The results were reported as the stirring number (SN). Duplicate measurements were taken. 2.7. The starch molecular size distribution The starch molecular size distribution was analyzed in duplicate using high performance size-exclusion chromatography with a refractive index detector (HPSEC-RI) following the method of

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Patindol and Wang (2003). Ten mg of each isolated starch was heated in 5 mL of 90% dimethyl sulfoxide (DMSO) for 1 h and then stirred at room temperature overnight. The sample was filtered through a 0.45 mm syringe filter and kept in a disposable tube for 30 min. Then, 200 mL of sample was injected into a HPSEC. The HPSEC system (Waters, Milford, MA) consisted of a 515 HPLC pump with an injector of 200 mL sample loop, an in-line degasser, a 2414 refractive index detector maintained at 40
C, and a Waters ultrahydrogel 250 (7.8  300 mm) column maintained at 55
C. The mobile phase of 0.1 M ammonium acetate with 0.02% sodium azide was eluted at a flow rate of 0.4 mL/min. The amylopectin and amylose contents in the starch samples were calculated automatically based on the areas of their corresponding peaks and expressed as percentage. 2.8. The amylopectin branch chain length distribution The amylopectin branch chain length distribution was determined in duplicate using high performance anion-exchange chromatography equipped with a pulsed amperometric detector (HPAEC-PAD) according to the method of Mendez-Montealvo et al. (2011). Ten mg of each isolated starch was stirred into 3.2 mL millipore water in a boiling water bath for 30 min. After cooling down, 0.4 mL of 0.1 M acetate buffer (pH 5.0) and 10 mL of isoamylase and 10 mL of pullulanase were added. The enzymatic reaction was carried out overnight in a water bath at 40
C with stirring at 150 rpm. Enzyme activity was terminated by adding 0.21 mL of 0.2 M NaOH to the mixture and then heating it in a boiling water bath for 15 min. The sample was cooled for 5 min and filtered through a 0.45 mm syringe filter (NYL w/GMF, Whatman, Clifton, NJ, USA). Then, 0.6 mL of supernatant was transferred into an auto sampler vial and injected into a HPAEC-PAD. The HPAECPAD (Dionex ICS-3000) system consisted of the following components: an ICS-3000 Dual pump, an ICS-3000 Eluent Organizer, an ICS-3000 Detector/Chromatography Module, a 4  50 mm CarboPac PA1 guard column, a 4  250 mm CarboPac PA1 analytical column, and an AS Auto sampler. The gradient system consisted of two eluents: eluent A (150 mM aqueous NaOH) and eluent B (500 mM NaNO3 in 150 mM aqueous NaOH). The amylopectin chain-length distributions were divided into four fractions depends on the degree of polymerization (DP) with ranges as follows: Achains (DP6-12), B1-chains (DP13-24), B2-chains (DP25-36), and B3-chains (DP37-60). The average chain length (CL) was calculated as the cumulative sum of the product of DP and the percentage of the relative areas for all the identified peaks. 2.9. Pasting properties Evaluation of pasting properties was performed in duplicate using the RVA according to Approved Method 61-02 (AACC, 2000). Rice starch (3 g, 12% moisture content) was dispersed in 25 ml distilled water in a RVA canister. The slurry was mixed at 960 rpm for 10 s to allow thorough dispersion; then the speed was reduced to 160 rpm for the remainder of the run. The heating and cooling cycles were programmed in the following manner. The slurry was held at 50
C for 1 min, heated to 95
C within 3.48 min and then held at 95
C for 2.7 min. It was subsequently cooled to 50
C within 3.88 min and then held at 50
C for 1.24 min, while maintaining a rotation speed of 160 rpm. The parameters recorded were peak time, pasting temperature, peak viscosity, trough viscosity, final viscosity, breakdown, and setback. 2.10. Statistical analysis The SPSS for Windows program (SPSS 11.0) was employed to

analyze the results. Data were subjected to analysis of variance (ANOVA). The differences among means were identified by Duncan's multiple-range test (DMRT) at significant differences of P < 0.05. 3. Results and discussion 3.1. Incubation time for pre-germination of paddy Table 1 shows the pre-germination time of paddy from three Thai rice cultivars. After the fixed soaking time of 12 h, the soaked paddy contained 30.5e33.5% moisture content, and the paddy which had a lower moisture content after soaking required a longer incubation time than those which had a higher moisture content. The three stages of EGL were obtained from paddy of the three rice cultivars after being incubated for different time periods. The soaked paddy from KDML105 which had the lowest moisture content after soaking required a longer incubation time to reach the three stages of pre-germination than other cultivars. This result agrees with previous work of Panchan and Naivikul (2009) who found that the typical moisture content for pre-germination of Indica paddy (Suphan Buri1, 25.12% amylose content) was at least 30%. However, in their study it took 14 h of soaking to reach 30% moisture content and another 14 h of incubation to reach the 1st stage of pre-germination. The difference in results may be due to the effects of the different rice cultivars. 3.2. Chemical composition and starch yield Chemical compositions and starch yields are shown in Table 2. The moisture contents of all starches samples were in the ranged of 9.5e10.7%, and UGBR starches had the highest starch yields. Generally, high purity rice starch contains low amounts of protein and lipid and the amount of lipids existing in rice is much lower than protein. Therefore, isolation of rice starch mainly involves removing protein, and the goal for the protein content of isolated milled rice starch is less than 0.5% (Suksomboon and Naivikul, 2006). In the present work, the starch yields of UGBR from the three rice cultivars were quite low, which was probably due to the other components initially present that can interfere with the starch extraction process. The starch yields of the three rice cultivars were reduced after pre-germination. The low starch yield after pre-germination was mainly due to the decomposition of native starch by hydrolytic enzymes (Xu et al., 2012). Besides the change in starch that occurred during pregermination, the protein content of starches from the three stages of PGBR was also affected by pre-germination. The crude protein contents that remained in PGBR starches may have resulted from a phenomenon that led to a subsequent increase in the initial protein content of PGBR. During pre-germination, the proteins were broken down to amino acids by activated enzymes. Some parts of these amino acids then entered various respiratory and carbon cycles and are utilized for the synthesis of proteins in the growing part of seedling (Palmiano and Juliano, 1972). This may be the reason why larger amount of crude protein content was remained in the PGBR starches than those of UGBR. Pre-germination also affected the amylose contents of low and high amylose rice cultivars, while the waxy one was not significantly (P > 0.05) affected. 3.3. a-amylase activity The SN indicates the changes in physical properties of the starch caused by a-amylase. As the enzyme activity increases, the SN decreases. In this study, the a-amylase activities of three-stage PGBR flour from all rice cultivars increased (SN decreased) during

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Table 1 Pre-germination durations of paddy from three Thai rice cultivars. Rice cultivar

RD6 KDML105 RD31

Pre-germination durations Moisture content (%)

Soaking time (h)

Incubation time (h)

Total pre-germination time (h)

1st stage

2nd stage

3rd stage

1st stage

2nd stage

3rd stage

33.5 30.5 32.8

12 12 12

16 24 20

20 32 24

24 36 32

28 36 32

32 44 36

36 48 44

Table 2 The chemical compositions, starch yields, aand stirring number (SN).b Rice cultivar

Pre-germination duration

Moisture content (%)

Starch yield (%db)

RD6

UGBR

10.5 ± 0.2c

30.8 ± 0.1a

KDML105

PGBR-1st PGBR-2nd PGBR-3rd UGBR

10.2 10.0 10.0 10.1

± ± ± ±

0.2d 0.0de 0.1e 0.2de

29.5 26.4 21.5 26.9

± ± ± ±

0.1c 0.1f 0.0i 0.0e

0.7 0.8 0.9 0.8

± ± ± ±

0.1g 0.0fg 0.0ef 0.0fg

0.2 0.2 0.2 0.2

± ± ± ±

0.0bc 0.1c 0.1c 0.1c

RD31

PGBR-1st PGBR-2nd PGBR-3rd UGBR

10.1 9.5 11.0 10.7

± ± ± ±

0.1de 0.1f 0.2a 0.1b

23.3 22.2 18.2 30.5

± ± ± ±

0.1g 0.0h 0.0l 0.1b

0.8 0.9 1.0 0.8

± ± ± ±

0.1f 0.0e 0.1d 0.1f

0.2 0.1 0.1 0.3

± ± ± ±

0.0c 0.1c 0.1c 0.1ab

PGBR-1st PGBR-2nd PGBR-3rd

9.6 ± 0.1f 10.7 ± 0.1b 9.6 ± 0.1f

28.6 ± 0.1d 20.4 ± 0.1j 20.1 ± 0.1k

Chemical composition (%db) Crude protein

Crude fat

0.7 ± 0.1g

0.1 ± 0.1c

1.2 ± 0.0c 1.3 ± 0.0b 1.5 ± 0.0a

SN

Amylose

0.4 ± 0.1a 0.5 ± 0.1a 0.4 ± 0.1a

6.5 ± 0.4f

2396b

6.4 6.4 6.2 17.4

± ± ± ±

0.2f 0.0f 0.0f 0.0d

262f 76f 47f 2939a

17.4 17.2 16.6 31.6

± ± ± ±

0.2d 0.2d 0.0e 0.1a

1543d 1531d 1085e 2987a

31.3 ± 0.0a 30.5 ± 0.5b 29.7 ± 0.3c

2777ab 2618ab 2011c

UGBR ¼ Ungerminated brown rice, PGBR ¼ Pre-germinated brown rice. a Means value ± SD with different small letters in the same column are significantly different (P < 0.05). b The stirring number (SN) was based on 14% moisture basis.

pre-germination as shown in Table 2. This result agrees with Charoenthaikij et al. (2009) who reported that a-amylase activities of waxy (RD6) and non-waxy (KDML105) brown rice increased when steeping time was increased from 24 to 48 h. Among three rice cultivars, the changes in a-amylase activities of three-stage PGBR flour from RD6 were greater than others. The a-amylase activities of PGBR flour were significantly (P < 0.05) different from UGBR flour at the 1st stage of pre-germination for RD6 and KDML105, but at the 3rd stage for RD31. The pre-germination durations had an impact on the a-amylase activities of three-stage PGBR flour from KDML105 and RD31. The highest a-amylase activities of PGBR flour from KDML105 and RD31 were found at the 3rd stage of pre-germination. It was found that the changes in aamylase activities led to the reduction of starch yields and AACs of PGBR starches. As the a-amylase activities increased, the starch yields and AACs of PGBR starches from KDML105 and RD31 decreased. However, the AACs of three-stage PGBR starches from RD6 remained unchanged. 3.4. Changes in starch molecular size distribution All starches samples showed differences in molecular size distributions as revealed by HPSEC-RI (Table 3). During the HPSEC-RI analysis, starch fractions from elution profiles were categorized into two fractions (amylopectin and amylose) on the basis of the retention time and the area of the peaks eluted which varies depending on molecular size. Isolated UGBR starch from RD6 had a significantly (P < 0.05) higher amylopectin content (93.02%), while RD31 had a significantly (P < 0.05) higher amylose content (18.58%) than others. Once pre-germination occurred, the amylose and amylopectin contents of PGBR starches isolated from KDML105 and RD31 were significantly (P < 0.05) different from their UGBR starches at the 1st

Table 3 Molecular size distributions of isolated UGBR and three-stage PGBR starches.a Rice cultivar

Pre-germination duration

Amylopectin (%)

RD6

UGBR

93.0 ± 0.1a

Amylose (%)

KDML105

PGBR-1st PGBR-2nd PGBR-3rd UGBR

93.0 93.7 93.9 83.9

± ± ± ±

0.1a 0.1a 0.5a 0.1e

7.0 6.3 6.6 15.5

± ± ± ±

0.1e 0.1e 0.2e 0.7b

RD31

PGBR-1st PGBR-2nd PGBR-3rd UGBR

87.7 88.2 89.0 81.4

± ± ± ±

0.0c 0.3bc 0.2b 0.4e

12.7 12.3 11.0 18.6

± ± ± ±

0.0c 0.4c 0.3d 0.4a

PGBR-1st PGBR-2nd PGBR-3rd

81.8 ± 1.0e 84.2 ± 0.6d 84.5 ± 0.4d

7.1 ± 0.0e

18.2 ± 1.0a 15.8 ± 0.6b 15.6 ± 0.4b

UGBR ¼ Ungerminated brown rice, PGBR ¼ Pre-germinated brown rice. a Means value ± SD with different small letters in the same column are significantly different (P < 0.05).

and 2nd stages of pre-germination, respectively, while both contents of RD6 remained unchanged. During pre-germination, the amylose and amylopectin molecular size distributions of KDML105 and RD31 showed a gradual decrease of amylose and a gradual increase of the amylopectin contents, indicating that amylose may gradually hydrolyze to a smaller molecular size which led to a decrease in the amylose content. These results imply that the changes during pre-germination between low and high amylose rice may contribute to the changes in amylose contents. Thus, RD6 which contains less amylose content than the others showed no significant change. However, these results were somewhat contradictory to the results of Mohan et al. (2010) who found that after brown Indica (Jaya) and Japonica (Yamadanishiki) rice were germinated up to

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from 69.81 to 65.45% after germination of long-grain brown rice, while the proportions of medium and long-chain amylopectin fractions (B2 and B3 chains) were slightly increased from 10.12 to 12.16%, and 20.07e22.39%, respectively. Researchers suggested that the degradation of starch during the germination process had some effects on the fine structures of amylopectin. The A and B1 chains of the amylopectin branch chains are the most external (exterior) and form double helices within the native granules. Thus, some of them were mainly degraded into oligosaccharides by hydrolytic enzymes, and utilized for energy for the growing plumules (Xu et al., 2012).

three days, the germinated brown Indica and Japonica rice starches showed a lower amylopectin content and a higher amylose content than UGBR starches. Researchers suggested that the slight decrease in the amylopectin content or slight increase in the amylose content could be due to the apparent increase in amylose content from the partial break down of the long branch chain of amylopectin. The difference in results may be due to the effects of the rice cultivar and germination conditions used in the experiments. The longer germination time used in Mohan et al. (2010) led to increase amylase activity and this could break down high molecular weight molecules more than would occur in the earlier stages of germination.

3.6. Pasting properties 3.5. The amylopectin branch chain length distribution The pasting properties of isolated UGBR and the three-stage PGBR starches are shown in Table 5. Isolated UGBR starch from RD6 provided a significantly (P < 0.05) lower peak viscosity (3360 cP) than the three-stage PGBR starches (3662e4240 cP). The changes in peak viscosities of isolated PGBR starches from RD6 occurred at the 1st stage of pregermination and it continued to increase until the 3rd stage. The peak viscosities of isolated UGBR starches from KDML105 (3686 cP) and RD31 (2159 cP) were significantly (P < 0.05) lower than their 2nd stage PGBR starches (3860 and 2279 cP). The results showed that the isolated UGBR starch from RD6 (1726 cP) had significantly (P < 0.05) lower breakdown viscosity than those of the three-stage PGBR starches (1838e2083 cP). The breakdown viscosities of isolated UGBR starches from KDML105 (1058 cP) and RD31 (502 cP) were significantly (P < 0.05) lower than their 2nd stage PGBR starches (1161 and 645 cP). The higher breakdown viscosities were related to the higher peak viscosities of PGBR starches. After cooling down to 50
C, both UGBR and the three-stage PGBR starches showed increased viscosities. The isolated UGBR starches from the three rice cultivars showed lower final and setback viscosities than those of the three-stage PGBR starches. The significant (P < 0.05) changes in setback viscosities occurred when the RD6 was incubated to the 3rd stage (293 cP) and the 1st stage for both KDML105 (2049 cP) and RD31 (1350 cP). The UGBR and the three-stage PGBR starches from RD6 showed significantly (P < 0.05) lower setback viscosities than those of KDML105 and RD31. The amylose content of rice starch was found to correlate positively with RVA setback viscosity (Noosuk et al., 2003). Thus, the higher amylose contents in low and high amylose rice cultivars were responsible for the higher

The amylopectin branch chain length distributions of all starches based on HPAEC-PAD are shown in Table 4. The amylopectin branch chains were classified into A (DP6-12), B1 (DP13-24), B2 (DP25-36), and B3 (DP37-60) on the basis of the number of glucose units per chain (Hizukuri, 1986). The amylopectin branch chains of isolated UGBR and the threestage PGBR starches from the three rice cultivars were composed of larger proportions of B1 chain followed by A chains, and the CL ranged at 17.6e20.1. The B1 to B3 amylopectin branch chains of UGBR starches differed significantly (P < 0.05) between different rice cultivars, while the A chains showed no significant (P > 0.05) difference. Isolated UGBR starch from RD6 had higher A chain (32.2% vs. 31.9%) and lower B3 chain (4.1% vs. 5.7%) than RD31. Once the three rice cultivars were pre-germinated, a decrease in A and B1 chains and an increase of B2 and B3 chains were found. At the 1st stage of pre-germination, the A and B1 chains from RD6 were decreased (32.2e25.4%, 53.5 to 50.3%), while B2 and B3 chains were increased (10.3e14.5%, 4.1e9.9%), respectively. It was found that the changes in amylopectin chain length distributions of RD6 were much more than other cultivars. As summarized in Table 3, RD6 waxy rice starch consists mainly of amylopectin, and the amylopectin contents were higher than others. Thus, the predominant changes in amylopectin fine structures of RD6 could be occurred probably due to the degradation of amylopectin molecules by activated a-amylase. The effects of pre-germination on amylopectin chain length distributions changes in these results are in agreement with the results of Xu et al. (2012) who reported a decreased short-chain amylopectin fractions (A and B1 chains)

Table 4 Amylopectin branch chain length distributions of isolated UGBR and three-stage PGBR starches.a Rice cultivar

Pre-germination duration

Amylopectin branch chain length distribution (%) DP6-12 (A-chains)

DP13-24 (B1-chains)

DP25-36 (B2-chains)

53.5 ± 0.1b

10.3 ± 0.0e

DP37-60 (B3-chains)

UGBR

32.2 ± 0.3a

KDML105

PGBR-1st PGBR-2nd PGBR-3rd UGBR

25.4 25.0 25.3 31.9

± ± ± ±

0.6e 0.1e 0.6e 0.2ab

50.3 50.5 50.6 55.0

± ± ± ±

0.0f 0.2f 0.1f 0.3a

14.5 14.5 14.2 8.9

± ± ± ±

0.2a 0.2a 0.4a 0.1g

9.9 10.0 9.9 4.5

± ± ± ±

0.4a 0.2a 0.3a 0.0f

20.1 20.1 20.1 17.6

± ± ± ±

0.2a 0.0a 0.1a 0.1f

RD31

PGBR-1st PGBR-2nd PGBR-3rd UGBR

29.5 29.3 29.2 31.9

± ± ± ±

0.0d 0.1d 0.0d 0.3ab

51.1 51.3 51.0 52.4

± ± ± ±

0.2de 0.1d 0.1e 0.2c

11.8 11.9 12.4 10.0

± ± ± ±

0.1c 0.0c 0.1b 0.0f

7.6 7.5 7.5 5.7

± ± ± ±

0.0b 0.0b 0.0b 0.0e

18.9 18.8 18.9 17.8

± ± ± ±

0.0b 0.0b 0.0b 0.0de

PGBR-1st PGBR-2nd PGBR-3rd

31.4 ± 0.1bc 30.9 ± 0.2c 30.8 ± 0.2c

52.4 ± 0.1c 52.3 ± 0.1c 52.1 ± 0.2c

10.3 ± 0.0ef 10.7 ± 0.0d 10.9 ± 0.0d

UGBR ¼ Ungerminated brown rice, PGBR ¼ Pre-germinated brown rice. DP ¼ Degree of polymerization as expressed as glucose unit. CL ¼ Average chain length. a Means value ± SD with different small letters in the same column are significantly different (P < 0.05).

4.1 ± 0.1g

CL

RD6

5.9 ± 0.0de 6.1 ± 0.1cd 6.2 ± 0.0c

17.8 ± 0.0e

18.0 ± 0.0cd 18.0 ± 0.1cd 18.0 ± 0.0c

H. Pinkaew et al. / Journal of Cereal Science 70 (2016) 116e122

121

Table 5 Pasting properties of isolated UGBR and three-stages PGBR starches.a Rice cultivar

Pre-germination duration

Peak time (min)

Pasting temp. (
C)

Viscosity (cP) Peak

Trough

Breakdown

Final

Setback

RD6

UGBR

3.7 (0.0)g

61.8 (0.0)e

3360 (36.6)e

1633 (20.4)fg

1726 (23.0)c

1847 (27.9)h

KDML105

PGBR-1st PGBR-2nd PGBR-3rd UGBR

3.7 3.7 3.5 6.6

(0.1)g (0.1)g (0.0)h (0.1)a

61.8 61.8 62.1 63.1

(0.0)e (0.0)e (0.4)e (1.1)d

3662 4043 4240 3686

(25.8)d (16.0)b (35.0)a (1.0)d

1823 2156 2156 2628

(13.7)e (11.0)d (41.9)d (69.0)c

1838 1886 2083 1058

(15.3)b (6.6)b (8.5)a (68.8)e

2089 2429 2445 4087

(16.8)g (14.9)f (32.6)f (34.0)c

266 272 293 1451

(11.8)gh (13.1)gh (14.5)g (40.8)d

RD31

PGBR-1st PGBR-2nd PGBR-3rd UGBR

6.4 6.4 6.3 6.1

(0.1)b (0.0)b (0.1)c (0.1)d

63.5 63.7 64.9 72.6

(0.4)d (0.8)d (0.0)c (0.4)b

3672 3860 3867 2159

(12.3)fd (12.0)c (65.4)c (4.2)h

2682 2698 2743 1656

(52.0)bc (61.8)ab (33.8)a (10.9)fg

990 1161 1125 502

(45.5)f (51.5)d (47.6)d (8.4)i

4738 4666 4908 2504

(17.2)b (47.3)b (23.2)a (35.4)f

2049 1954 2141 835

(42.1)b (29.4)c (101.0)a (72.5)f

PGBR-1st PGBR-2nd PGBR-3rd

6.1 (0.1)d 6.0 (0.1)e 5.8 (0.0)f

72.8 (1.2)b 73.4 (1.1)b 73.6 (0.1)a

2197 (1.8)h 2279 (9.4)g 2387 (27.1)f

1689 (24.9)f 1633 (24.6)fg 1601 (49.7)g

508 (23.2)i 645 (17.5)h 786 (24.6)g

2966 (66.8)e 3602 (107.8)d 3537 (114.1)d

217 (8.9)h

1350 (241.9)e 1950 (185.4)c 1937 (168.7)c

UGBR ¼ Ungerminated brown rice, PGBR ¼ Pre-germinated brown rice. a Means value (SD) with different small letters in the same column are significantly different (P < 0.05).

setback compared to the waxy rice cultivar. The pasting properties of native rice starch have been predominantly influenced by amylose content and the amylopectin branch chain length distribution. Waxy rice starch consists mainly of amylopectin with an absence of amylose-lipid complexes, and thus the starch granule swells rapidly (lower pasting temperature) and greatly (higher peak viscosity) (Jane et al., 1999). The isolated starch from the long-grained rice cultivar (27.2% amylose content) had the highest pasting temperature, final and setback viscosities, whereas isolated starch from the glutinous rice cultivar showed the highest peak and breakdown viscosities (Chung et al., 2011). Starch consisting of amylopectin with high A chains was reported to display a low peak and high breakdown viscosities since the short branch chains cannot provide a strong interaction to maintain the integrity of the swollen granules (Srichuwong and Jane, 2007). In the present work, the changes in pasting properties of PGBR starches were probably due to the changes of amylose contents and the amylopectin fine structures. After rice was pre-germinated, the decrease of amylose content was due to a-amylase activated (Table 2) during pre-germination and the low amylose content could contribute to the high peak viscosities of PGBR starches. In addition, the reduction of short A chains amylopectin branch chains (Table 4) in PGBR starches could be the other reason. Because amylopectin is the major component of rice starches, variations in the amylopectin fine structure could contribute to pasting property differences in the PGBR starches. As mentioned earlier, starch containing high proportions of A chain amylopectin displays a lower peak viscosity (Srichuwong and Jane, 2007). It can be concluded that the increase in peak viscosities of PGBR starches may be the result of a decrease in A chains of amylopectin. These results agree with the previous work of Musa et al. (2011) who reported amylose contents (23.83%, 21.78%) and peak viscosities (324 RVU, 328 RVU) of ungerminated and germinated Malaysian brown rice starches, respectively. However, in the study of Wu et al. (2013) there were no obvious changes in the viscosities during the first two days of germination, but significant (P < 0.05) increased in the viscosities were observed after three days of germination for medium and high amylose rice cultivars. The results of the present study are in contradiction to the results of Xu et al. (2012) who reported that after germination the peak viscosity of GBR starch was significantly (P < 0.05) lower than that of UGBR starch. The difference in results may be due to the different rice cultivars and types of rice: brown rice vs. the paddy used in the present experiments.

4. Conclusions The results indicated that the paddy which had a lower moisture content after soaking required a longer incubation time than those which had a higher moisture content. Increasing pre-germination durations caused an increase in a-amylase activities. The rice cultivars and the durations of pre-germination had some effects on changes in the starch yields, chemical compositions, physicochemical properties, and fine structures of isolated PGBR starches. Pre-germination led to lower starch yields of three rice starches and the reduction of AACs of low and high amylose rice cultivars. The starch degradation due to activated a-amylase during pregermination was also observed through changes in the amylopectin and amylose contents. An increase of the amylopectin content and decrease of the amylose content were observed after pregermination of KDML105 and RD31. However, the amylopectin and amylose contents of RD6 remained constant during pregermination. Significant changes in amylopectin branch chain length distributions were found especially for the A chains amylopectin of RD6. A decrease in A chains and increase in B3 chains were found after pre-germination of the three rice cultivars resulting in an increase in pasting temperatures and peak viscosities for the PGBR starches. Therefore, pre-germination could be used as a bio-modification method for modifying rice starches. Some of the quality characteristics of PGBR from the three rice cultivars can be controlled by pre-germination duration and the PGBR can be used as a raw material for specific foods. Acknowledgments This research was supported by the Thailand Research Fund (TRF) through the Royal Golden Jubilee Ph.D. program (contract No. PHD/0238/2551). References AACC, 2000. Approved Methods of the AACC, 10th ed. American Association of Cereal Chemists, St. Paul, MN. Agboola, S., Ng, D., Mills, D., 2005. Characterisation and functional properties of Australian rice protein isolates. J. Cereal Sci. 41, 283e290. Charoenthaikij, P., Jangchud, K., Jangchud, A., Piyachomkwan, K., Tungtrakul, P., Prinyawiwatkul, W., 2009. Germination conditions affect physicochemical properties of germinated brown rice flour. J. Food Sci. 74 (29), C658eC665. Chung, H.J., Liu, Q., Lee, L., Wei, D., 2011. Relationship between structure, physicochemical properties and in vitro digestibility of rice starches with different amylose contents. Food Hydrocoll. 25, 968e975. Hizukuri, S., 1986. Polymodal distribution of the chain lengths of amylopectin, and its significance. Carbohydr. Res. 147, 342e347.

122

H. Pinkaew et al. / Journal of Cereal Science 70 (2016) 116e122

Jane, J., Chen, Y.Y., Lee, L.F., McPherson, A.E., Wong, K.S., Radosavljevic, M., Kasemsuwan, T., 1999. Effect of amylopectin branch chain length and amylose content on the gelatinization and pasting properties of starch. Cereal Chem. 76 (5), 629e637. Juliano, B.O., 1971. A simplified assay for milled-rice amylose. Cereal Sci. Today 16, 334. Kaneko, M., Itoh, H., Ueguchi-Tanaka, M., Ashikari, M., Matsuoka, M., 2002. The aamylase induction in endosperm during rice seed germination is caused by gibberellins synthesized in epithelium. Plant Physiol. 128, 1264e1270. Mendez-Montealvo, G., Wang, Y.-J., Campbell, M., 2011. Thermal and rheological properties of granular waxy maize mutant starches after b-amylase modification. Carbohydr. Polym. 83, 1106e1111. Mohan, B.H., Malleshi, N.G., Koseki, T., 2010. Physico-chemical characteristics and non-starch polysaccharide contents of Indica and Japonica brown rice and their malts. LWT-Food Sci. Technol. 43 (5), 784e791. Musa, A.S.N., Umar, I.M., Ismail, M., 2011. Physicochemical properties of germinated brown rice (Oryza sativa L.) starch. Afr. J. Biotechnol. 10 (33), 6281e6291. Noosuk, P., Hill, S.E., Pradipasena, P., Mitchell, J.R., 2003. Structure-viscosity relationships for Thai rice starches. Starch/St€ arke 55, 337e344. Palmiano, E.P., Juliano, B.O., 1972. Biochemical changes in the rice grain during

germination. Plant Physiol. 49, 751e756. Panchan, K., Naivikul, O., 2009. Effect of pre-germination and parboiling on brown rice properties. Asian J. Food Agro-Industry 2 (04), 515e524. Patindol, J., Wang, Y.-J., 2003. Fine structures and physicochemical properties of starches from chalky and translucent rice kernels. J. Agric. Food Chem. 51, 2777e2784. Pinkaew, H., Naivikul, O., 2012. Effect of pre-germination process on chemical and physicochemical properties of Thai waxy rice flour and starch. Cereal Food World 57 (4), A65eA66. Srichuwong, S., Jane, J., 2007. Physicochemical properties of starch affected by molecular composition and structure: a review. Food Sci. Biotechnol. 16 (5), 663e674. Suksomboon, A., Naivikul, O., 2006. Effect of dry- and wet-milling processes on chemical, physicochemical properties and starch molecular structures of rice starches. Kasetsart J. Nat. Sci. 40, 125e134. Wu, F., Chen, H., Yang, N., Wang, J., Duan, X., Jin, Z., Xu, X., 2013. Effect of germination time on physicochemical properties of brown rice flour and starch from different rice cultivars. J. Cereal Sci. 58, 263e271. Xu, J., Zhang, H., Guo, X., Qian, H., 2012. The impact of germination on the characteristics of brown rice flour and starch. J. Sci. Food Agric. 92, 380e387.