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. 2023 Aug 28;33(5):1113–1122. doi: 10.1007/s10068-023-01411-0

Characteristics of amylose–lipid complex prepared from pullulanase-treated rice and wheat flour

Sang Hoon Lee 1, Wen Yan Huang 1, Jinhee Hwang 1, Hyeock Yoon 2, Wan Heo 3, Jiyoun Hong 1,2, Mi Jeong Kim 1, Chang-Soo Kang 4, Bok Kyung Han 1,2,✉, Young Jun Kim 1,2,✉
PMCID: PMC10908976  PMID: 38440677

Abstract

This study aimed to evaluate the properties of amylose–lipid complexes in rice and wheat flours utilizing pullulanase as a debranching enzyme. Rice and flour were both treated with pullulanase before being combined with free fatty acids to form compounds denoted as RPF (rice-pullulanase-fatty acid) and FPF (flour-pullulanase-fatty acid), respectively. Our results showed that RPF and FPF had higher complex index and lower hydrolysis values than enzyme-untreated amylose–lipid complexes. Furthermore, RPF and FPF demonstrated lower swelling power and higher water solubility values, indicating changes in the physical properties of the starches. In vivo studies showed that RPF and FPF caused a smaller increase in blood glucose levels than untreated rice and flour, highlighting their potential use as functional food ingredients. These findings provide valuable information for the development of novel rice-and wheat-based foods with improved nutritional and physiological properties.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-023-01411-0.

Keywords: Resistant starch, Rice, Flour, Oleic acid, Pullulanase

Introduction

Starch, an important carbohydrate found in different plants such as cereals, tubers, and roots, is essential in determining the quality of various food products (Najafi et al., 2016). The two polymers of starch, amylose and amylopectin, have different structural characteristics. Amylopectin consists of highly branched chains of glucose units linked by α-1,4 glycosidic bonds with α-1,6 glycosidic bonds every 20–21 units. Amylose, in contrast, is a linear polymer with α-1,4 linkages that can be up to 270 units long and is mostly organized in helical structures (Tester et al., 2004). Because of the properties of its α-1,4 linkages, amylose can form amylose–lipid complexes or V-amylose when inclusion complexes with lipids are formed. The ability to establish hydrogen bonds and the degree of hydrophobicity of the ligand are critical factors in the formation of these complexes (Kang et al., 2020). Although there have been studies on the formation of amylose–lipid complexes, it is still unclear whether they only occur with α-glucan crystals.

Pullulanase, a debranching enzyme, hydrolyzes α-1,6 glycosidic bonds in amylopectin, resulting in an increase in free linear molecules (Miao et al., 2009). Several studies, including one by Zhao and Lin (2009), have reported that debranching treatment can generate more linear chain molecules, which increases mobility between molecules and facilitates the formation of ordered crystalline structures. Enzymatic starch modification generates a large number of appropriately polymerized linear chains, promoting complexation reactions between starch and guest molecules (Reddy et al., 2019). After hydrolyzing starches from various botanical sources with pullulanase, the debranched starches showed higher complexing ability and solubility than native starches. Furthermore, pullulanase debranching is reported to be more effective than other methods of forming amylose–lipid complexes (Wang et al., 2020).

Amylose–lipid complexes are formed by the non-covalent inclusion of lipids in an amylose-helix-shaped structure (Zhang et al., 2020). Depending on the size of the monoacyl chain, the helix typically consists of three turns around each monoacyl chain, with each turn containing six to eight glucosyl residues (Li et al., 2021). This complex is formed by the gelatinization of starch in the presence of lipids. The transformation of amylose from its random coil structure to a helix shape with altered glycosidic bond angles increases resistance to digestion by limiting its accessibility to digestive enzymes in resistant starch type 5 (RS-5) starch-lipid complexes (French and Murphy, 1977). The complex alters the performance of starchy foods by impeding gelatinization progression and retarding retrogradation, which changes their pasting properties (Chen et al., 2017), creating novel resistant starches, acting as a vehicle to protect and deliver sensitive ligands such as unsaturated fatty acids (Lesmes et al., 2009), and being used in starch-based films (Liu et al., 2016). Therefore, the amylose–lipid complexes alter the physical and chemical properties of starch, influencing its solubility, swelling capacity, viscosity, and absorption capacity.

Oleic acid (OA) is a long-chain fatty acid with a double bond. It exhibits higher solubility than saturated fatty acids and is more likely to form complexes with amylose (Ai et al., 2013). In this study, OA was used as a model long-chain fatty acid to prepare starch-lipid complexes that had high resistant starch content using currently available processing conditions. Furthermore, to date, there have been no reports investigating the characteristics of starch with increased amylose content and enhanced resistant starch through the combination of lipid with pullulanase-treated starch. Therefore, the objective of our study is to develop resistant starch with increased amylose content through the combination of a lipid with pullulanase-treated starch and evaluate its properties to enhances RS-5 formation. We studied the effects of digestion on these complexes and found that pullulanase-treated starch-lipid complexes digested more slowly than untreated starch, potentially providing health benefits. The results of this study provide valuable insights into the formation of starch-lipid complexes during food processing and may help manipulate food product quality.

Materials and methods

Materials

Commonly consumed refined rice (R) and flour (F) were purchased from Ottogi (Seoul, Korea) and Samyang Co. (Seoul, Korea), respectively. Ilshin Wells (Cheongju, Korea) supplied the following lipids: free fatty acids (FFA; OA 95%, other lipids 5%), monoglycerides (MG; OA 95%, other lipids 5%), diglycerides (DG; OA 95%, other lipids 5%), and triglycerides (TG; OA 95%, other lipids 5%). Pullulanase (Promozyme D2, 1350 NPUN/g) was purchased from Novozymes (Bagsvaerd, Denmark). Ingredion International (Bridgewater, NJ, USA) supplied a type 2 resistant starch product derived from high-amylose maize containing approximately 60% RS and 40% amylopectin (Hi-Maize® 260).

Preparation of gelatinized starch with or without added lipids

Polished white rice was used in this study, and it was ground to obtain the rice starch used in the research. Gelatinized starch samples were prepared as follows: 5.0 g each of ground rice starch and flour with or without added lipids (10% w/w, 0.5 g) were gelatinized in a water bath (~ 95 °C) for 8 min under constant manual operation (Ai et al., 2013). During gelatinization, four different types of lipids (10% w/w, 0.5 g) were added to the starch in deionized water (× 5, w/w, dry solid basis), and the mixtures were continuously stirred while heating until the starch was completely gelatinized.

Preparation of pullulanase-treated starch with or without lipids

To facilitate gelatinization, rice starch and flour suspensions (10%, w/w dry basis) were placed in a boiling water bath at 100 °C for 10 min. The slurry (20%, w/w, dsb in diluted pH 4.5 buffer solution) was incubated in a water bath at 60 °C. Pullulanase at various concentrations (25, 50, 75, and 100 PUN/g dry starch) was used to induce starch debranching. The starch solution was stirred continuously at 450 rpm for 48 h. A representative sample was then heated to 100 °C to inactivate the enzyme, freeze-dried, and finely ground to a 100-mesh size (Demirkesen-Bicak et al., 2018). Freeze-dried samples of the enzyme-treated rice starch and flour were combined with the lipids (10% w/w, 0.5 g) in a water bath.

Hydrolysis rate analysis

We used the Megazyme resistant starch assay kit to determine the resistant starch fractions as previously described Ma et al. (2021). Briefly, starch samples were incubated at 37 °C with a mixture of amylo-1,6-glucosidase and α-amylase to mimic mammalian starch digestion. After 20 min, a 0.5 mL aliquot of the digest was removed and vigorously mixed with 3.5 mL of 50% ethanol. The mixture was centrifuged at 1500×g for 15 min. The supernatants were decanted, and the glucose content was measured using the glucose oxidase/peroxidase reagent. The readily digestible starch (RDS) content was calculated by multiplying the measured glucose by 0.9 and then dividing by 100. The amount of glucose produced after 120 min of digestion was determined using a similar procedure. The slowly digestible starch (SDS) content was defined as the difference in the glucose released between 20 and 120 min. The degree of hydrolysis was calculated by adding the RDS and SDS content.

Complex index of starch-fatty acid complexes

The complex index of starch-lipid complexes was determined using a modified version of the procedure described by Liu et al. (2016). The starch-lipid complex samples (0.3 g, dsb) were weighed and placed in centrifuge tubes. Deionized water was then added to achieve a total weight of 5 g. The suspensions were completely gelatinized by heating the tubes in a boiling water bath for 10 min. After cooling to 25 °C, the gelatinized starch samples were mixed with 25 mL of deionized water, vortexed for 2 min, and centrifuged at 4000 rpm for 15 min. A 0.5 mL aliquot from each supernatant was mixed with 15 mL of deionized water and 2 mL of iodine solution (2.0% KI and 1.3% I2 in deionized water, w/v). Starch samples without FA were used as controls. The absorbance of the samples was measured at 690 nm using an A560 UV–Vis spectrophotometer (SPECTROstar Nano; BMG LABTECH Corp., Ortenberg, Germany).

Amylose contents analysis

Apparent amylose content (AAC) was determined using the method described by Juliano et al. (1981) with minor modifications. Starch flour (100 mg) was accurately weighed and placed in a 100 mL measuring flask, and 1 mL of 95% ethanol was added to each sample in triplicate. Subsequently, 9 mL of 1 M NaOH was added, and the mixture was gently shaken to dissolve any major lumps. The resulting solution was boiled until it became clear. After cooling at to 25 °C, distilled water was added to the starch solution to fill the flask to the 100 mL mark. Next, 500 μL of the sample solution was pipetted into a 15 mL centrifugal tube, and 200 μL of I2 (0.2%)/KI (2%) solution, 100 μL of acetic acid (1 mol/L), and 9 mL of distilled water were added to the tube and mixed thoroughly. This solution was allowed to stand at 25 °C for 10 min before absorbance was measured using a spectrophotometer (LABTECH Corp., Ortenberg, Germany). A standard curve was prepared using purified potato amylose (A0512; Sigma-Aldrich, St. Louis, MO, USA) to calculate the AAC of each sample (Ma et al., 2021).

X-ray diffraction

The crystalline patterns of the starch-lipid complexes were analyzed using a Theta-type X-ray diffractometer (D2 PHASER, Bruker-AXS, Karlsruhe, Germany). The rice starch group (R) used in the experiment was polished white rice, which was ground into a powder for use. To determine the crystalline pattern, the instrument (set to 40 kV and 40 mA) measured the diffraction angle (2θ) between 4 and 40° (Liu et al., 2016).

Pasting properties

The pasting properties of the isolated starch were analyzed using a rapid viscosity analyzer (Newport Scientific, Sydney, Australia). A starch suspension weighing 28.0 g and containing 8% starch (w/w, dsb) was equilibrated at 50 °C for 1 min, then heated to 95 °C at a rate of 6 °C/min, held at 95 °C for 5 min, and finally cooled to 50 °C at the same rate. The paddle was rotated at 160 rpm, except for the first 10 s when the rotation was 960 rpm. The analysis was performed in duplicate (Keppler et al., 2018).

Swelling power analysis

One gram of starch was accurately weighed and corrected for moisture to obtain the dry weight (A). The sample was mixed with 40 mL water in a 50 mL centrifuge tube containing a small stirrer magnet. The tube was then placed in a 92.5 °C water bath equipped with a thermo-regulator and a magnetic stirrer set at 100 rpm for 30 min. The samples were centrifuged for 20 min at 2000×g after cooling to 20 °C in a cool water bath with continuous gentle magnetic stirring for 30 min (water changed after 5, 10, and 20 min). The supernatant was removed by suction, and the weight of the residue (B) was measured to calculate the total starch swelling power (Chen et al., 2017).

Starch swelling power = weight of residue (B)/dry weight of starch (A).

Water solubility

The water-holding capacity of the samples was determined using the method of Liu et al. (2015) with slight modifications. Briefly, a 5% (w/v) aqueous suspension of the sample was heated in a water bath at 85 °C for 1 h with constant stirring. The suspension was then centrifuged at 8000×g for 10 min. The supernatant was then carefully removed, and the residue paste was weighed. Water-holding capacity was calculated as the weight of the residue paste per gram of dry sample.

Oral starch tolerance test

All animal care and experimental procedures were approved by the Ethics Committee of Korea University (approval number: KUIACUC-2020-0043). All institutional and national guidelines for the care and use of laboratory animals were followed. Male C57BL/6 J mice, aged 6 weeks and weighing 18–20 g, were obtained from Raon Bio Inc. (Yong-in, Korea). The mice were held in a controlled environment free of specific pathogens with a 12-h light/dark cycle, temperature maintained between 22 and 25 °C, and relative humidity between 50 and 60%. After fasting the mice for more than 12 h, blood was collected from the caudal vein, and fasting blood sugar levels were measured using a blood glucometer (Autocheck, Diatech Korea Co., Ltd.). There were six experimental groups, each consisting of five animals: the 0.5% carboxymethylcellulose sodium salt only (CMC, C5678, Sigma-Aldrich, St. Louis, MO, USA)-treated group (vehicle), the Hi-maize group (Hi, positive control), the rice starch group (R), the OA-treated rice starch group after pullulanase treatment (RPF), the wheat flour group (F), and the wheat flour group treated with OA after pullulanase treatment (FPF). For each group, starch was dissolved in 0.5% carboxymethylcellulose sodium salt (C5678, Sigma-Aldrich, St. Louis, MO, USA) at 3 g/kg body weight and administered to the mice. Blood glucose was measured at 15-min intervals for up to 240 min after administering starch, and the area under the curve (AUC) was calculated to determine the change in blood glucose (Kanti Bera et al., 2014).

Statistical analysis

Data are expressed as mean ± standard deviation (SD) or standard error (SE). Analysis of variance (ANOVA) was used for statistical analysis, followed by Duncan’s multiple range test. All statistical analyses were performed using the SPSS software (Version 25, IBM, NY, USA), and statistical significance was set at p < 0.05.

Results and discussion

Analysis of complex index values and digestibility of starch-lipid complexes in rice starch and flour

The complex index values of the rice starch- and flour-lipid complexes are presented in Fig. 1A and B. There are significant differences in the complex index values depending on the type of lipid incorporated into the rice starch or flour. The rice-lipid complexes showed the highest complex index value for rice starch-free fatty acid (RFA) complexes (33.88%), followed by rice starch-monoglyceride (RMG) (20.56%), rice starch-diglyceride (RDG) (7.37%), and rice starch-triglyceride (RTG) (6.30%) complexes (Fig. 1A). The flour-lipid complexes followed a pattern similar to that of the rice-lipid complexes (Fig. 1B). The highest complex index value was found in flour-free fatty acid (FFA) complexes (80.56%), followed by flour-monoglyceride (FMG) (68.79%), flour-diglyceride (FDG) (28.96%), and flour-triglyceride (FTG) (19.28%) complexes (Fig. 1B). The results revealed that the complex index value decreased as the degree of free fatty acid binding to glycerol increased in both rice starch and flour, depending on the type of lipid used. These findings are consistent with previous research on the wheat starch-diglyceride and -triglyceride systems (Chao et al., 2018). Another study found that starch complex formation occurred with monoglycerides but not with glycerol alone during heat treatment (Bhatnagar and Hanna, 1994). Furthermore, prior to pullulanase treatment, the amylose contents of rice starch and flour were 17.97% and 25.59%, respectively, and this content increased in a dose-dependent manner as the pullulanase concentration increased (Fig. S1A and B). The observed trend in the complex index values, higher values in wheat flour than in rice starch, may be because of the difference in amylose content between the two samples (Fig. 1A and B).

Fig. 1.

Fig. 1

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The effect of synthesis conditions on the complex index and enzyme digestibility of starch-lipid complexes. (A, B) Complex index values synthesized using different types of lipids with rice (A) and flour (B) starches. (C, D) Enzyme digestibility measured for rice (C) and flour (D)-derived samples. R native rice starch, RFA rice starch-free fatty acid complex, RMG, rice starch-monoglyceride complex, RDG rice starch-diglyceride complex, RTG rice starch-triglyceride complex, F native flour starch, FFA flour starch-free fatty acid complex, FMG flour starch-monoglyceride complex, FDG flour starch-diglyceride complex, FTG flour starch-triglyceride complex. Data are expressed as mean ± SD. Means in the same column with different letters are significantly different (p < 0.05)

To evaluate the digestibility of starch-lipid complexes, the enzyme hydrolysis content was calculated based on the glucose content after the enzyme reaction. The enzymatic hydrolysis of RFA and FFA was significantly lower than that of the other rice starch-lipid and flour-lipid complexes, respectively (Fig. 1C and D). A previous study found that forming a starch-fatty acid complex can reduce susceptibility to enzymatic degradation (Li et al., 2021). Our findings indicated that both rice starch and flour exhibited the highest formation of starch-lipid complexes in the presence of free fatty acids. Furthermore, higher oil solubility and a greater amount of a nonpolar backbone, such as glycerol, inhibited the formation of starch-lipid complexes (Li et al., 2020). The reason for this is that glycerol possesses a small molecular structure and lacks the essential characteristics needed for adequate interactions with amylose, thus preventing its binding (Li et al., 2021). Consequently, starch-lipid complexes containing free fatty acids but no glycerol were selected for further investigation.

Effect of pullulanase treatment on starch-lipid complex formation and digestibility in rice starch and flour

The use of pullulanase to break the 1,6-bonds in rice starch and flour starch is a well-known method to increase amylose content (Wang et al., 2020). In this study, rice starch and flour samples were treated with various concentrations of pullulanase, ranging from 0 to 100 PUN/g, and then analyzed for changes in their amylose content (Fig. S1). The results revealed a concentration-dependent increase in amylose content up to 50 PUN/g but no significant difference between 75 and 100 PUN/g when compared to 50 PUN/g (Fig. S1). Consequently, the optimal pullulanase concentration was determined to be 50 PUN/g.

Rice starch showed a noteworthy increase in the complex index of rice starch lipids after treatment with 50 PUN/g pullulanase, as shown in Fig. 2A. Similarly, after enzyme treatment, the complex formation of flour-lipids increased from 80.57 to 93.21% (Fig. 2B). Significantly higher complex formation was observed in the group in which amylose content increased due to pullulanase treatment. Prior to enzyme treatment, the degree of amylose complexation in rice starch was 6.1% (18.0 × 0.339); however, it increased to 31.0% (32.7 × 0.948) after enzyme treatment (Table S1). Similarly, before enzyme treatment, the degree of amylose complexation in flour was 25.6% (28.6 × 0.897), indicating that 25.6% of the amylose was bound to free fatty acids. After enzyme treatment, the degree of complexation in flour increased to 38.8% (41.6 × 0.932), indicating that 38.8% of the amylose was bound to free fatty acids (Table S1). This confirmed that the increase in the amylose content of starch through pullulanase treatment led to an increase in amylose–lipid complex formation. The formation of an amylose–lipid complex may account for the decrease in the hydrolysis rate, as this complex is more resistant to digestion by α-amylase and amyloglucosidase than free amylose molecules (Li et al., 2020).

Fig. 2.

Fig. 2

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Properties of enzyme-modified starch-lipid complexes. (A, B) CI values synthesized from pullulanase (50 PUN/g)-treated rice starch (A)-or flour starch (B)-lipid complex samples. (C, D) Enzyme digestibility measured for rice (C)-and flour (D)-derived samples. R native rice starch, RFA rice starch-free fatty acid complex, RPF pullulanase-treated rice starch-free fatty acid complex, F native flour starch, FFA flour starch-free fatty acid complex, FPF pullulanase-treated flour starch-free fatty acid complex. Data are expressed as mean ± SD. Means in the same column with different letters are significantly different (p < 0.05)

The digestibility of starch-lipid complexes was then assessed by analyzing the hydrolysis of rice starch and flour starches with or without pullulanase treatment, followed by the incorporation of free fatty acids. The hydrolysis rates of R, RFA, and RPF were 72.48%, 59.92%, and 43.27%, respectively (Fig. 2C). Flour (F) had a hydrolysis value of 67.58%, whereas FFA and FPF had values of 61.11% and 38.12%, respectively (Fig. 2D). The hydrolysis values of the RPF and FPF groups treated with pullulanase were significantly lower than those of the untreated starches (Fig. 2C and D). Free fatty acid complexation with rice starch and flour significantly decreased both RDS and SDS content (Fig. 2C and D), indicating lower enzyme accessibility due to the formation of amylose–lipid complexes (Hasjim et al., 2013) These findings are consistent with previous research, indicating that increased amylose content leads to a corresponding increase in amylose–lipid complexes and decreased hydrolysis values (Li et al., 2021). Furthermore, amylose is structurally more resistant than amylopectin to starch-degrading enzymes, resulting in lower hydrolysis values as the amylose concentration increases. In conclusion, after enzymatic treatment with pullulanase, both rice starch and flour showed an increase in RS-5 content.

The effects of amylose–lipid complexes on swelling power and water solubility of starch

Starch granules are primarily composed of unstructured amylose, which is released during gelatinization as the amylopectin component swells (Liu et al., 2023). Rice starch had a swelling power of 12.86, whereas RFA and RPF had values of 8.20, and 6.75, respectively (Fig. 3A). The reduced swelling power of RFA and RPF could be attributed to the formation of an insoluble film by amylose–lipid complexes, which reduces their water-binding capacity (Chen et al., 2017). Similar swelling powers were observed in flour, with F, FFA, and FPF exhibiting values of 12.75, 9.42, and 3.30, respectively (Fig. 3B). The reduced swelling power of RPF and FPF compared with that of RFA and FFA can also be attributed to the formation of an insoluble film, leading to a reduced water-binding capacity. The patterns observed in flour were similar to those in rice starch and may be attributed to the formation of an insoluble film by amylose-free fatty acid complexes (Xu et al., 2012). Additionally, the lower levels of amylopectin present in RPF and FPF contributed to their reduced swelling power. The presence of hydroxyl groups on amylose also results in lower absorption and swelling power owing to the formation of amylose-free fatty acid complexes and crystallites (Manimegalai and Parimalavalli, 2023).

Fig. 3.

Fig. 3

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Swelling power and water solubility of starch-lipid complex. (A, B) Swelling power measured for rice (A)-and flour (B)-derived samples. (C, D) Water solubility measured for rice (C)-and flour (D)-derived samples. R native rice starch, RFA rice starch-free fatty acid complex, RPF pullulanase-treated rice starch-free fatty acid complex, F native flour starch, FFA flour starch-free fatty acid complex, FPF pullulanase-treated flour starch-free fatty acid complex. Data are expressed as mean ± SD. Means in the same column with different letters are significantly different (p < 0.05)

Water solubility refers to the soluble portion of starch that cannot absorb moisture (Guraya et al., 2001). The water solubilities of R, RFA, and RPF were 8.23, 9.13, and 11.63, respectively (Fig. 3C), whereas those of F, FFA, and FPF, were 8.33, 9.21, and 13.30, respectively (Fig. 3D). Significantly higher values were observed for RPF and FPF than for RFA or FPF alone. These results imply that the portion of pullulanase-degraded amylose starch that did not bind to free-fatty acids contributed to increased water solubility. Shorter linear chains of starch, which are more likely to align and aggregate, may have led to chain aggregation and gel network formation through hydrogen bonding and hydrophobic interactions (Liu et al., 2015). Furthermore, due to the high water solubility of starch granules, they are used in the production of starch-based films (Kim et al., 2015). These results emphasize the potential applications of starch-based materials in the food industry.

Evaluating pasting and viscosity profiles of different starches

Figure 4 displays the rapid viscosity analysis profiles for various starch systems, from which parameters such as pasting temperature, peak viscosity, trough viscosity, setback, and final viscosity were derived (Keppler et al., 2018). Peak viscosity values for R and F were 1023 and 1402 cp, respectively, with final viscosity values of 1660 and 1806 cp, and trough viscosity values of 765 and 828 cp, respectively (Fig. 4A and B). The pullulanase-treated starches showed minimal changes in viscosity, even under thermal conditions, particulary in relation to setback viscosity (final viscosity-trough viscosity) (Fig. 4A and B). The reason was that the formation of amylose–lipid complexes creates a barrier that prevents water from penetrating the starch molecules, leading to a decrease in viscosity (Liu et al., 2023). The setback viscosity was influenced by the final viscosity, which is related to the aging properties and increases as the setback value increases (Zaidul et al., 2007). Partial modification of proteins within the starch and swelling of the starch are known to cause an increase in its viscosity peak (Van Steertegem et al., 2013). The RPF and FPF developed in this study had similar initial values for final viscosity, indicating that they were resistant to retrogradation and were less affected by the proteins present in the starch than R and F. Consequently, these starches were less influenced by molecular bonding because their viscosities did not change significantly (Heo et al., 2015).

Fig. 4.

Fig. 4

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Rapid viscosity profile of enzyme-treated starch-lipid complexes. (A) Samples derived from rice. (B) Samples derived from flour. R native rice starch, RPF pullulanase-treated rice starch-free fatty acid complex, F native flour starch, FPF pullulanase-treated flour starch-free fatty acid complex

X-ray diffraction analysis reveals unique crystallinity patterns of starches

Starches have degrees of association and crystallinity as well as distinctive wavelengths that can be determined using X-ray diffraction pattern analysis. Starches can be classified as A, B, and V-types using X-ray diffraction, with A-type starches having an amylopectin glucose chain of 23–29 units. Examples of A-type starch include rice starch and flour. Figure 5 depicts the crystallinity patterns of the rice starch and flour samples dried under different conditions. Peaks were observed in R and F at 10.1°, 11.2°, 15.2°, 15.3°, 17.2°, 18.1°, 22.7°, and 23.0° (Fig. 5A and B), with peaks at 15.2° and 18.1° indicating the characteristics of A-type starch (Junka and Rattanamechaiskul, 2022). However, peaks at 12.0° and 12.5° were observed for RFA and FFA (Fig. 5A and B), respectively, indicating a transition from A-type to V-type crystallinity. When starch granules break, amylose is released and reacts with the fatty acids present in the starch to form an amylose-fatty acid complex (Ma et al., 2021). RPF and FPF showed strong peaks at 11–12° and 20° (Fig. 5A and B), respectively, which have previously been reported to represent peak values for amylose–lipid complex binding. As the complexity increases, V-type crystallinity also increases (Zhang et al., 2020). The calculated crystallinities (%) of RPF and FPF, as determined by X-ray diffraction, were significantly higher than those of the other groups (Table S2). The relative crystallinity values in RPF and FPF were also significantly higher, indicating an increase in crystallinity owing to the high number of amylose–lipid complex bonds (Wang et al., 2020). Thus, these results confirm that pullulanase-treated R and F combine with free fatty acids to form highly crystalline V-type starch.

Fig. 5.

Fig. 5

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X-ray diffraction patterns of native starch, starch-lipid, and enzyme-modified starch-lipid complexes. (A) Samples derived from rice. (B) Samples derived from flour. R native rice starch, RFA rice starch-free fatty acid complex, RPF pullulanase-treated rice starch-free fatty acid complex, F native flour starch, FFA flour starch-free fatty acid complex, FPF pullulanase-treated flour starch-free fatty acid complex

Resistant starches RPF and FPF show potential for improving glycemic control

Glucose is the primary factor responsible for postprandial metabolic and endocrine changes (Kanti Bera et al., 2014). When compared to regular starch, the formation of amylose–lipid complexes can reduce the in vivo digestion of starch into glucose (Li et al., 2021). To investigate the effects of amylose–lipid complexes on postprandial hyperglycemia, an oral starch tolerance test (OSTT) was performed. Hi-maize (Hi), a resistant starch type 2 with a high amylose content that is known to cause lower increases in blood glucose, served as a positive control (Deng et al., 2010). When compared to the R group, mice fed with RPF showed an improvement in both the rate and extent of glycemic reduction, with the AUC decreasing by 26% (Fig. 6A and B). The OSTT results of the RPF group were comparable to those of the Hi group. No statistically difference was observed in the OSTT of mice fed with FPF and those fed with F (Fig. 6A and B).

Fig. 6.

Fig. 6

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Effect of RPF and FPF on glycemic control. (A, B) Oral starch tolerance test (3 g/kg) following overnight fasting (> 12 h) (A) in 7-week-old male C57BL/6 J mice and area under the curve (AUC) (B). (C) Changes in blood glucose levels from 0 to 30 min after sample administration. Vehicle mice administrated with 3% CMC solution, Hi mice administrated with hi-maize, R mice administrated with rice starch, RPF mice administrated with pullulanase-treated rice starch-fatty acid complex, F mice administrated with flour starch, FPF mice administrated with pullulanase-treated flour starch-free fatty acid complex. All mice were fed via gavage. One-way ANOVA was used for all comparisons except for (A). Two-way ANOVA was used specifically for OSTT (A) to determine statistical significance. *p < 0.05, **p < 0.01, ***p < 0.005 (vs. Hi). Data are expressed as mean ± SEM. Means in the same column with different letters are significantly different (p < 0.05)

Notably, the highest blood glucose levels were observed 30 min after the sample was administered. However, at the 30-min mark after sample administration, both RPF and FPF groups had significantly lower blood glucose levels than R and F groups, with values comparable to the Hi group (Fig. 6C). The breakdown of RDS is responsible for elevated post-meal blood glucose levels. For example, digestive enzymes typically break down the starch component, amylopectin, within 20 min, releasing glucose into the bloodstream within 30 min (Benmoussa et al., 2007). In contrast, resistant starches, such as Hi, cannot be broken down by digestive enzymes, resulting in a reduction in the blood glucose spikes. Our findings suggest that RPF and FPF, as novel resistant starches, may modulate postprandial blood glucose levels, similar to existing resistant starches. These results underscore the potential use of RPF and FPF as functional food ingredients to improve glycemic control.

This study investigated the effect of different lipid types on the formation and in vitro and in vivo digestibility of starch-lipid complexes. The results showed that free fatty acids were the most effective in producing resistant starch complexes. Furthermore, pullulanase-treated starches combined with free fatty acids (RPF or FPF) formed more complexes than are found in regular rice starch and wheat starches. Moreover, this study revealed that RPF and FPF are novel resistant starches with increased crystallinity, lower viscosity, and improved glycemic control. Finally, the findings of this study provide important insights into the development of novel rice starch- and wheat-based food products that provide nutritional and physiological benefits.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 89 KB) (88.3KB, docx)

Acknowledgements

This work was supported by a grant (Graduate School Education Program of Regulatory Sciences for Functional Food, 21153MFDS604) from the Ministry of Food and Drug Safety of the Republic of Korea. This research was supported by the BK21 FOUR (Fostering Outstanding Universities for Research) funded by the Ministry of Education (MOE, Korea) and National Research Foundation of Korea (NRF).

Declarations

Conflict of interest

The authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this study.

Footnotes

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Contributor Information

Bok Kyung Han, Email: hanmoo@korea.ac.kr.

Young Jun Kim, Email: yk46@korea.ac.kr.

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