http://orcid.org/0000-0001-9354-3087
ABSTRACT
We here characterized 27 japonica rice cultivars grown in Heilongjiang province and evaluated the relationship among their iodine absorption curve, physical properties, and ratio of 13 kDa prolamin. We developed the novel estimation formulae for ratio of 13 kDa prolamin and overall hardness (H2) with the use of Aλmax and λmax.
China is the largest rice-producing country, accounting for 32% of the global production from 20% of the global rice-growing regions. Japonica rice production in Heilongjiang province, a semi-arid region in northeast of China, accounted for 25% of the total production in China in 2000s. This province produces early – maturing, flavorful, high-yielding rice varieties that were developed by crossbreeding Japanese varieties with indigenous varieties [Citation1].
Rice grain accumulates starch and protein during ripening. The amylose contents of starch and the protein content are the main factors that determine the taste of rice [Citation2]. In general, the application of fertilizers does not affect the protein composition during ripening stage [Citation3]. It is necessary to clarify relationship between molecular-biological characterization of protein and cultivation conditions for rice good taste. We here characterized 13 japonica rice cultivars and 14 crossbreeding japonica lines in Heilongjiang province and evaluated the relationship among their iodine absorption curve, apparent amylose content (AAC), physical properties, and ratio of 13 kDa prolamin.
Samples of 13 japonica rice cultivars (Longdao 18, Longdao 16, Longjing 31, Longjing 46, Longdao 21, Suijing 18, Longjing 3, Suijing 15, Songjing 22, Songjing 16, Hajing 2, Longken 201 and Daohuaxiang 4) and 14 crossbred japonica lines (CJ-6, CJ-7, CJ-10, 2017B6351, 2017C2718, 2017C2728, 2017C2737, 2017C2740, 2017C2765, 2017C2857, 2017C2815, 2017C2819, 2017C2865, 2017C2895) grown in Heilongjiang province were provided by Heilongjiang Food Quality and Safety Research Center. The AAC comprises a large amount of amylose and small amount of super-long chains (SLC) of amylopectin. We estimated the AAC of milled rice using the iodine colorimetric method as previously described [Citation4]. The iodine absorption spectrum [Citation5] was analyzed from 200–900 nm. Low – amylose rice becomes soft and sticky after cooking, while high – amylose rice is harder, with the grains remaining separated [Citation6]. We observed that the AAC values were higher in the 13 japonica rice cultivars (range, 10.7%–15.4%; mean, 13.0%) than in the 14 crossbred japonica lines (range, 10.0%–14.2%; mean, 12.5%) (Supplemental Table 1). The molecular structures of many starches, including the molecular sizes of amylose and the amylopectin branch chain lengths, have been reported previously [Citation7,Citation8]. The high – molecular – weight amyloses tend to have a longer λmax. The λmax values of the 13 japonica rice cultivars (range, 573.8–595.3 nm; mean, 584.4 nm) were slightly higher than those of the 14 crossbred japonica lines (range, 572.7–590.2 nm; mean, 582.0 nm) (Table 1). The Aλmax values of the 13 japonica rice cultivars (range, 0.271–0.320; mean, 0.296) were slightly higher than those of the 14 crossbred japonica lines (range, 0.257–0.308; mean, 0.290) (Supplemental Table 1).
The starches in the rice cultivars grown under low temperatures have significantly higher amylose content and lower SLC amylopectin content than cultivars grown under high temperatures [Citation9–Citation11]. We observed that λmax/Aλmax was lower in the 13 japonica rice cultivars than in the 14 crossbred japonica lines (Supplemental Table 1).
Protein content was determined using a near-infrared spectrometer (AN-820, Kett Co., Ltd, Tokyo). It is well known that protein contents affect the eating qualities of cooked rice grains [Citation2] and especially, prolamin deteriorates eating quality of rice [Citation12]. SDS-PAGE was conducted using a 12% polyacrylamide gel according to the method described by Laemmli [Citation13] with a slight modification. The protein concentrations were calculated based on the intensities of various bands on the gel after SDS-PAGE analysis as determined using the ATTO densitography software library (CS Analyzer ver 3.0) (Supplemental Figure 1). PB-I is highly enriched in prolamin and constitutes approximately 20% of the milled rice protein content. Prolamin comprises three polypeptide subunits with molecular masses of 10, 13, and 16 kDa. PB-II contains primarily glutelins and constitutes 60–65% of the milled rice protein [Citation14,Citation15].
The hardness and stickiness of the boiled rice grains were measured using a Tensipresser (My Boy System, Taketomo Electric Co., Tokyo, Japan) with the individual grain method for low compression (25%) and high compression (90%) tests [Citation16]. As shown in supplemental Table 1, we observed that the hardness of the surface layer (H1) was higher in the 13 japonica rice cultivars (range, 673.5–878.9 ×10−5 N/cm2; mean, 770.2 × 10−5 N/cm2) than the 14 crossbred japonica lines (range, 573.9–951.6 × 10−5 N/cm2; mean, 760.8 × 10−5 N/cm2), and the hardness of the overall layer (H2) of the 13 japonica rice cultivars (range, 16,141.7 – 20,956.8 × 10−5 N/cm2; mean = 19,285.9 × 10−5 N/cm2) was higher than that of the 14 crossbred japonica lines (range, 15,778.9 – 20,790.1 × 10−5 N/cm2; mean = 18,528.3 × 10−5 N/cm2). The stickiness of the surface layer (–H1) was higher in the 13 japonica rice cultivars (range,–117.386 – 69.411 × 10−5 N/cm2; mean = –100.136 × 10−5 N/cm2) than the 14 crossbred japonica lines (range,–107.971 – 45.415 × 10−5 N/cm2; mean = –77.909 × 10−5 N/cm2), and the stickiness of overall layer (–H2) was higher in the 13 japonica rice cultivars (range,–3527.5 – 2440.9 × 10−5 N/cm2; mean = –3173.1 × 10−5 N/cm2) than in the14 crossbred japonica lines (range,–3700.0 – 2550.7 × 10−5 N/cm2; mean = –3121.9 × 10−5 N/cm2).
Among the 13 japonica rice cultivars, the ratios of 13 kDa prolamin correlated positively with the AAC (r = 0.82), λmax (r = 0.76), Aλmax (r = 0.82), Fb3 (DP ≥ 37: the proportion of longer chains than 37DP among the amylopectin molecules) (r = 0.82; p < 0.01), and overall hardness (H2) (r = 0.62; p < 0.05) and negatively with λmax/Aλmax (r = −0.82; p < 0.01) and surface adherence (L3) (r = −0.58; p < 0.05) (Supplemental Table 2A). Furthermore, those of the 14 crossbred japonica lines showed a similar tendency (Supplemental Table 2B). In addition, the overall hardness (H2) correlated positively with AAC (r = 0.81), λmax (r = 0.82), Aλmax (r = 0.79), Fb3 (DP ≥ 37) (r = 0.79; p < 0.01), but negatively with λmax/Aλmax (r = −0.78; p < 0.01); those of the 14 crossbred japonica varieties showed a similar tendency (Supplemental Table 2B).
As the close relationship was shown between prolamin and indices by iodine colorimetric analysis in supplemental Table 2, we estimated the ratios of 13 kDa prolamin in rice grains based on the iodine absorption curve obtained for milled rice. The equation, which had a multiple coefficient of determination of 0.67 based on the calibration curve ()), is follows: Ratios of 13 kDa prolamin = 157.931 × Aλmax – 28.407. Validation test of the formula with unknown samples showed that a multiple regression coefficient of 0.54 was obtained with the application of the above – mentioned formula to 14 crossbred japonica lines in Heilongjiang province ()). We tried to estimate the overall hardness (H2) of the cooked rice grains based on the iodine absorption curve obtained for milled rice ()). The equation had a multiple coefficient of determination of 0.68 based on the calibration curve. We obtained the following formula for estimating the overall hardness (H2) using the 13 japonica rice cultivars; Overall hardness (H2) = 15.85 × λmax – 7294. ) shows that a multiple regression coefficient of 0.86 was obtained with the application of this formula to the 14 crossbred japonica lines indicating that these equations can be applied to the crossbred japonica varieties. This finding shows that we can evaluate the characteristic physical properties of cooked rice of various cultivars using this simple, low-cost spectroscopic method.
Figure 1. Formula for estimating ratios of 13 kDa prolamin from the iodine absorption curve of milled rice flour.
(a). Ratios of 13 kDa prolamin = 157.931 × Aλmax – 28.407. The equation had a multiple regression coefficient of 0.67 based on the calibration. (1, Longdao 18; 2, Longdao 16; 3, Longjing 31; 4, Longjing 46; 5, Longdao 21; 6, Suijing 18; 7, Longjing 3; 8, Suijing 15; 9, Songjing 22; 10, Songjing 16; 11, Hajing 2; 12, Longken 201; 13, Daohuaxiang 4). (b) Examination estimation formula with unknown samples. A multiple regression coefficient of 0.54 was obtained by applying the above formula to the 14 crossbred japonica lines. (1, CJ-6; 2, CJ-7; 3, CJ-10; 4, 2017B6351; 5, 2017C2718; 6, 2017C2728; 7, 2017C2737; 8, 2017C2740; 9, 2017C2765; 10, 2017C2857; 11, 2017C2815; 12, 2017C2819; 13, 2017C2865; 14, 2017C2895).
Figure 2. Formula for estimating the overall hardness (H2) from the iodine absorption curve of milled rice flour.
(a). Overall hardness (H2) of overall = 15.848 × λmax – 7294.336. The equation had a multiple regression coefficient of 0.68 based on the calibration. (1, Longdao 18; 2, Longdao 16; 3, Longjing 31; 4, Longjing 46; 5, Longdao 21; 6, Suijing 18; 7, Longjing 3; 8, Suijing 15; 9, Songjing 22; 10, Songjing 16; 11, Hajing 2; 12, Longken 201; 13, Daohuaxiang 4). (b) Examination estimation formula with unknown samples. A multiple regression coefficient of 0.86 was obtained by applying the above formula to the 14 crossbred japonica lines. (1, CJ-6; 2, CJ-7; 3, CJ-10; 4, 2017B6351; 5, 2017C2718; 6, 2017C2728; 7, 2017C2737; 8, 2017C2740; 9, 2017C2765; 10, 2017C2857; 11, 2017C2815; 12, 2017C2819; 13, 2017C2865; 14, 2017C2895).
Authors contribution
Ohtsubo K and Kawano M designed the experiments; Nakamura S, Li H, Dai C, and Zhang R performed experiments; Nakamura S, and Ohtsubo K wrote the paper.
NakamuraNoteSupplemental_table_fig_0123.pdf
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We express gratitude to the students working in our laboratory for their help in conducting the experiments.
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No potential conflict of interest was reported by the authors.
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