Effect of inhibition of different SBE(s) on amylose content
SBEs are responsible for the synthesis of α-1,6-glucosidic linkages by catalysing the cleavage and transfer of α-1,4-linked glucan chains to branch 6-hydroxyl groups, thus producing branches in amylopectin. Reduction in SBEs’ activity reduces the frequency of branch points in the amylopectin fraction and increases the AC (Morell et al., 2004). Results from the present study showed that the AC in transgenic lines with p13dsSBE1 (SBEI RNAi) showed no difference compared with the WT, whereas the AC was increased in transgenic lines with p13dsSBE3 (SBEIIb RNAi), especially with p13aSBE13 (antisense for both SBEI and SBEIIb). Our results suggest that different SBEs play different roles in starch biosynthesis in plants. In rice endosperm, SBEI is the major enzyme, which accounts for about 60-70% of the total SBEs’ activity, and SBEIIa and SBEIIb contribute equally to the rest of the SBEs’ activity (Yamanouchi and Nakamura, 1992). In potato tubers, Jobling et al. (1999) reported that SBEA, the minor form of SBE, has a major impact on starch structure. SBEA can complement the activity of SBEB, and these two SBEs interact with each other. AC was increased to 77–78% when both SBEA and SBEB were inhibited to below 1% of the wild-type activities (Schwall et al., 2000). Safford et al. (1998) reported no significant differences in AC or amylopectin branch length profiles of transgenic tuber starches in potato with anti-SBEB, whereas starches from plants with anti-SBEA showed an apparent increased AC of 38% versus 30% in controls (Jobling et al., 1999). In contrast to potato and rice, inhibition of single SBEA in maize ae mutant and pea r mutant produced very high-amylose starches in these plants (Shannon and Garwood, 1984; Wang et al., 1998), suggesting that the roles of SBEs are different in different plants.
In this study, AC was increased to about 65% by the inhibition of both SBEI and SBEIIb. Further research is needed to determine whether AC can be further increased in rice by inhibiting SBEIIa as well as SBEI and SBEIIb. In addition, it is worthwhile to investigate whether alternative approaches such as using a mutagen (e.g. ethyl methane sulphonate), targeting induced local lesions in genomes technique, inhibiting other enzymes (e.g. soluble starch synthases), enhancing GBSS, or a combination of inhibiting soluble starch synthesis and enhancing GBSS can lead to a very high-amylose starch in rice.
Starch makes up approximately 80%–90% of the dry weight of rice grains, in which amylopectin is normally one of the main components. Reducing or shutting down the biosynthesis of amylopectin is likely to affect the level of starch accumulating in endosperm and thus affects kernel weight. In this study, the kernel weight of high-amylose rice was significantly decreased (approximately 38%, P < 0.01). The appearance of the transgenic kernel was opaque and smaller than the control (Figure 3a,d). The width and thickness of the transgenic kernel were significantly decreased (P < 0.01), thus reducing the kernel weight (Table S1). SEM revealed that the starch granule in the transgenic rice had more irregular shapes, so they packed less tightly in the endosperm and created air spaces, which decreased the kernel weight. The dry weights of rice kernels at different filling stages from HA2 rice and WT rice were determined (Figure S4). The kernel weights of the HA2 rice and the WT differed after on the eighth day after flowering (DAF). In addition, HA2 rice kernels displayed growth stagnation at 12 DAF, whereas the kernel weight for the WT increased sharply from 8 to 12 DAF and a little more from 12 to 20 DAF. Therefore, the kernel weight difference between HA2 rice and the WT mainly depended on difference in the growth on the 8–20 DAF.
Effects of increased AC on RS and enzyme resistance
In addition to the increase in AC, RS and TDF levels were significantly increased (P < 0.01) in high-amylose rice (Figure 2e) (Zhu et al., 2011). Our GPC (Zhu et al., 2011) and HPAEC results (Figure 4) showed more long chains in the high-amylose rice amylopectin. Those long chains have the ability to form more stable double helices with reduced enzyme susceptibility, resulting in higher RS and TDF than in the WT amylopectin.
High-amylose rice showed more resistance to enzyme hydrolysis than the WT, not only because of its higher proportion of amylose and long amylopectin chains, but also because of its semicompound starch granules (Wei et al., 2010; Zhu et al., 2011). Compound starch granules consist of many individual granules held together by unknown forces and have less specific surface area than individual granules. Thus, compound granules bind less amylase than individual granules, which restricts hydrolysis.
High-amylose rice also displayed higher thermal resistance in DSC results than the WT. The DSC endotherm of HA2 rice starch was broader than the WT and showed a higher onset, peak and conclusion gelatinization temperatures. This agreed with results for maize starches with different ACs (Shi et al., 1998). High-amylose rice showed higher resistance on the pasting property and alkaline gelatinization, which confirmed the RS characteristic.
Potential health benefits of high-amylose rice
The feeding test on rats showed that the final body weight of the HA2 group was significantly lower (P < 0.05) than that of the WT group, even though total food intake did not differ. Those results indicate that high-amylose rice, rich in RS, may play a role in effective weight management. RS can help control body weight because it is a functional dietary fibre that can deliver some of the benefits of insoluble and soluble fibre (Premavalli et al., 2006; Bassaganya-Riera et al., 2011; Fuentes-Zaragoza et al., 2011). An increase in either soluble or insoluble fibre intake appears to increase postmeal satiety and decrease subsequent hunger (Howarth et al., 2001). In our feeding study on high-amylose rice, we found no significant effect of HA2 feeding on organ weight or the length of the colon (Table 1). Kim et al. (2003) also reported that the length of the small intestines, caecum, colon and rectum and the tissue weight of the caecum were not affected by feeding RSs from corn or rice. Notably, the caecum volume with its contents was larger in the HA2 group than in the WT group. The weight of the caecum with its contents for the HA2 group was 3.21 ± 1.26 g versus 2.81 ± 0.59 g for the WT group, which might be because RS escapes digestion in the small intestine and passes directly into the caecum where bacterial fermentation begins to occur, which enlarges the caecum volume. This phenomenon, of course, corroborated the definition of RS. No abnormal tissue changes were found in organ tissue section slides for rats fed the HA2 rice, indicating that high-amylose rice has no effect on animal organs.
Rats fed with HA2 and WT for 4 weeks showed no statistical significance in resting levels of plasma glucose, insulin, triglycerides or cholesterol, but the HA2 group showed elevated potassium. The plasma glucose in rats fed with HA2 was somewhat elevated compared with the rats fed with WT, presumably because of the slow release feature of RS as it was still being metabolized 16 h after consumption, whereas the WT rice starch was digested quickly (Aparicio-Saguilán et al., 2007). A positive correlation between the insulin and glucose levels was observed, which is also called the glucose/insulin response. Potassium absorption was significantly greater (P < 0.01) in rats fed with HA2 than in the WT. Several explanations for this effect can be proposed. Lopez et al. (2001), who found that the apparent Ca, Mg, Zn, Fe and Cu absorptions in rats were enhanced by raw potato starch and high-amylose cornstarch, indicated that an increase in the exchange area (enlargement of caecum and longer transit time) and the elevation of the caecal blood flow could be a reason. Moreover, the reduced pH in the caecum increases the solubility of these minerals. Schulz et al. (1993) also suggested that native RS raised Ca and Mg absorption because it tended to enhance the solubility of these minerals in ileal and caecal digests. Many reports in the literature indicate that the consumption of RS significantly reduces blood triglycerides and total cholesterol (Brites et al., 2011); however, the present study showed no statistical difference between rats fed with HA2 and WT, although a decreasing trend was found in the HA2 group. Significance might be found if more HA2 was added to the diet or if the rats were fed for a longer time.
RS helps to keep colon tissue healthy by producing protective compounds called SCFA, primarily composed of acetic, propionic and butyric acids, which reduce intestinal pH, encourage the growth of healthy bacteria in the bowel and discourage the growth of potentially harmful bacteria (Fuentes-Zaragoza et al., 2011; Regmi et al., 2011). The formation of butyrate seems to be especially beneficial, because it is the primary energy source for colon cells and also provides protection against colorectal cancer (Scharlau et al., 2009). In this study, the HA2 group produced significantly more SCFA (P < 0.01) and faecal matter (P < 0.05) and resulted in lower pH (P < 0.01) than the WT group. The butyrate level was doubled compared with the WT group. Those data indicate that a diet containing high-amylose rice improves gut health in rats.
Published research has shown that consumption of RS by humans resulted in a decreased glycaemic response in healthy individuals (Vonk et al., 2000) and diabetics (Giacco et al., 1998). High-amylose rice, HA2, in this study seems effective on the glucose response in type-2 diabetes, but no effect was found on normal SD rats or type-1 diabetic rats within 3 h.
In conclusion, rice with approximately 65% AC was developed; the high-amylose rice had high RS and TDF content. For the first time, we demonstrated that the diet containing the high-amylose rice had significant positive effects on animal health, such as a significantly reduced glycaemic response, an increased faecal output, increased faecal moisture, increased SCFA concentrations and decreased faecal pH. In addition, rats fed the WT rice gained twice as much weight in 4 weeks as those fed the HA2 diet, even though feed intake was equal. The relative weights of organs were not affected. This novel rice with its high AC, RS and TDF offers potential benefits for its use in foods and in medical and industrial applications.