Different multi-scale structural features of oat resistant starch prepared by ultrasound combined enzymatic hydrolysis affect its digestive properties

Graphical abstract

Abstract

In this research, oat resistant starch (ORS) was prepared by autoclaving-retrogradation cycle (ORS-A), enzymatic hydrolysis (ORS-B), and ultrasound combined enzymatic hydrolysis (ORS-C). Differences in their structural features, physicochemical properties and digestive properties were studied. Results of particle size distribution, XRD, DSC, FTIR, SEM and in vitro digestion showed that ORS-C was a B + C-crystal, and ORS-C had a larger particle size, the smallest span value, the highest relative crystallinity, the most ordered and stable double helix structure, the roughest surface shape and strongest digestion resistance compared to ORS-A and ORS-B. Correlation analysis revealed that the digestion resistance of ORS-C was strongly positively correlated with RS content, amylose content, relative crystallinity and absorption peak intensity ratio of 1047/1022 cm⁻¹ (R_1047/1022), and weakly positively correlated with average particle size. These results provided theoretical support for the application of ORS-C with strong digestion resistance prepared by ultrasound combined enzymatic hydrolysis in the low GI food application.

1. Introduction

Resistant starch (RS) is a general term for starch and its degradation products that cannot be digested and absorbed by the small intestine of healthy humans [1]. Generally, it can reach the colon after 2 h of consumption, and interacts with gut microbiota, such as promoting the growth of probiotics and producing short-chain fatty acids [2]. As a new type of dietary fiber, RS has similar physiological functions to dietary fiber, and without the shortcomings of poor taste and flavor of dietary fiber. In previous reports, it has been shown that RS has physiological functions, such as preventing colon cancer, lowering blood sugar, lowering cholesterol and inhibiting fat accumulation [3]. Due to excessive intake of diets such as sugars and animal fats, people are at increased risk of obesity and diabetes. Therefore, people are looking for foods that can lower blood sugar, lose weight, and have a low glycemic index (GI). Studies have shown that an increased proportion of whole grains in people's diets is associated with a reduced risk of obesity and diabetes. This is mainly due to the abundance of indigestible carbohydrates in whole grains, such as non-starch polysaccharides and resistant starches. In addition, the special structure of RS can improve its physical and chemical properties such as swelling power, viscosity, transparency and water holding capacity [4]. Therefore, RS has good development prospects and advantages for the functional food processing industry.

Oat is an annual herbaceous plant of the Poaceae family, which is divided into two types: husk oat and naked oat [5]. Oats have received more and more attention due to their high nutritional value and health benefits in preventing obesity and diabetes [6]. Most oats are eaten as whole grain products such as oatmeal, porridge, whole grain bread [7]. At present, the research on oat mainly focuses on functional components such as protein, dietary fiber, unsaturated fatty acid and β-glucan [8]. The content of starch in oat can reach up to 65%, but due to the poor regeneration stability, low transparency and high gelatinization temperature of native starch, its food application range is narrow [7]. The molecular structure of oat starch was changed by different methods to improve its physicochemical properties and increase the content of resistant starch. This is of great significance for improving the application of oat starch and its resistant starch in low GI foods and other functional foods.

However, the relationship between the structure of oat-resistant starch prepared by different methods and its digestion resistance was rarely studied [9]. The commonly used methods for preparing resistant starch include autoclaving-retrogradation cycle method, ultrasound method, enzymatic hydrolysis method, etc [10]. The autoclaving-retrogradation cycle method belongs to the physical method, which destroys the starch particle structure during the autothermal gelatinization process, rearranges its starch chain molecules, then promotes the formation of RS during the cooling regeneration process, and the product safety is high. Ultrasound technology is a new type of green processing technology, which can improve mass transfer and reaction efficiency due to its unique mechanical effect and cavitation effect. Ultrasound combined enzymatic hydrolysis could increase the contact frequency of substrate and enzyme, improve the efficiency of enzymatic hydrolysis reaction, promote the formation of RS, reduce its digestibility, and thus reduce the GI value of RS. There is currently no comprehensive information on the structural and anti-digestion properties of ORS prepared by ultrasound combined enzymatic hydrolysis. The specific purpose of this study was to prepare highly digestible resistant oat starch by autoclaving-retrogradation cycle, enzymatic hydrolysis and ultrasound combined enzymatic hydrolysis. Its physicochemical properties, structure and digestive properties were determined, and correlation analysis was carried out. This lays a foundation for studying the relationship between ORS structure and digestion resistance, which can promote the application of oat resistant starch in the preparation of low GI foods.

2. Materials and methods

2.1. Materials

Oat flour was obtained from Walmart Supermarket of Shanghai, China. Pullulanase (1200 npun/mL) was purchased from Sigma Chemical Co. (St. Louis, USA). The total starch, resistant starch and amylose/amylopectin assay kits were purchased from Megazyme International Ireland Limited, Ireland. All other chemicals and reagents were analytical grade.

2.2. Starch extraction

Oat starch (OS) was isolated according to the method described by Shah et al. with some modifications [11]. The oat flour and distilled water were mixed at a ratio of 1:8 (w/v). The pH was adjusted to 10 with NaOH (2 mol/L), and the experiment was carried out in a water bath at 40 °C for 3 h with continuous stirring during the reaction. Then it was passed through a 100-mesh sieve, and the filtrate was centrifuged at 4000 g for 10 min. The brown upper layer was scraped off, and the lower white part was washed three times with distilled water then dried at 40 °C in an oven.

2.3. Preparation of resistant starch from oat starch

Oat resistant starch ORS-A was prepared by autoclaving-retrogradation cycle method described by Shah et al. with some modifications [12]. The oat starch and distilled water were mixed at a ratio of 1:5 (w/v), and were autoclaved at 121 °C for 30 min. The starch was then cooled to room temperature and stored at 4 °C for 24 h. The autoclaving-retrogradation cycle was then repeated one more time. Finally, the samples were dried at 40 °C in an oven, then crushed and sieved to obtain oat resistant starch ORS-A.

Oat resistant starch ORS-B was produced by enzymatic hydrolysis method according to Liang et al. with some modifications [13]. The oat starch and distilled water were mixed at a ratio of 1:20 (w/v), then heated in a boiling water bath with constant stirring for 20 min. After the starch solution was cooled to 50 °C, the pH was adjusted to 5.0 with 0.05 M HCl. Pullulanase (27.5 npun/g starch) was added to the starch solution, then reacted at 50 °C for 12 h. After enzymolysis, the mixture was heated in a boiling water bath for 10 min, then cooled to room temperature and stored at 4 °C for 24 h. Finally, the samples were dried at 40 °C in an oven, then crushed and sieved to obtain oat resistant starch ORS-B.

After the pullulanase was added to the oat starch solution, the mixture was sonicated for 10 min at 240 W, and then enzymatically hydrolyzed at 50 °C for 12 h. The next steps were unchanged to obtain oat resistant starch ORS-C.

2.4. Determination of resistant starch and amylose content

The RS content was determined using the AOAC official method 2002.02, which has been described by Ren et al. [5]. The contents of total starch and amylose/amylopectin were measured with total starch assay kit and amylose/amylopectin assay kit (Megazyme International, Ireland, Ltd), respectively. Other basic components were carried out by following the AOAC protocol (AOAC, 2000).

2.5. Solubility and swelling power

The solubility and swelling power of starch samples were determined according to the method described by Liu et al. with some modifications [14]. M1 g starch sample was added a certain amount of distilled water to obtain 2% (w/v) starch solution. Then, the mixtures were heated at 30 °C, 50 °C and 90 °C with continuous stirring for 30 min. After the samples were cooled to room temperature, they were centrifuged at 4000 g for 10 min. The supernatant was collected, dried at 105 °C and weighed, recorded as M2 g. The sediment mass was recorded as M3 g.

Solubility (%) = 100 × M2/M1.

Swelling power (g/g) = M3 / (M1-M2).

2.6. Transparency and water holding capacity

100 mL of starch solution (1 %, w/v) was heated and stirred in a boiling water bath for 30 min, and then cooled to room temperature. A cuvette and an UV spectrophotometer were used to detect the transmittance of the starch solution at a wavelength of 620 nm, with distilled water as a blank control.

A starch sample (0.4 g, W1) was mixed with 15 mL of distilled water. The starch solution was incubated at 37 °C for 1 h and then centrifuged at 3000 g for 10 min. The supernatant was discarded and the sediment mass W2 was weighed.

Water holding capacity (%) = 100 × W2/W1.

2.7. Granule morphology

The Granule morphology of the starch samples were determined using a Hitachi Gemini 300 scanning electron microscopy (SEM, Hitachi High-Technologies, Tokyo, Japan) at magnifications of 200 × and 2000 × at an accelerator potential of 1.0 kV. Before observation, the samples were fixed on an aluminum plate using double-sided tape, and then a gold film was sprayed under vacuum.

2.8. Particle size distribution

The particle size distribution of the starch samples was measured by a Mastersizer 2000 (Malvern Instruments, Malvern, UK). 250 mg of the sample was added to 50 mL of distilled water, and the particle size distribution was measured after mixing. The refractive indices of starch samples and distilled water were set to 1.52 and 1.33, respectively. The particle size parameters of the samples: D [3,4] volume average particle size, D [2,3] surface area average particle size and Dv [10], Dv [48], Dv [90] (the maximum particle size of starch granules of this percentage 10%, 50%, and 90% in the starch system) were calculated and recorded using the installed software.

2.9. Thermal properties

The thermal properties of the starch samples were studied with a differential scanning calorimeter (DSC, TA Q2000 Instruments, New Castle, DE, USA). The dried starch sample (3.0 mg) was weighed into a platinum pan and distilled water (6 µL) was added. The pans were kept at room temperature for 1 h before analysis. Then the pans were heated at 10 °C/min from 20 °C to 200 °C. An empty platinum pan was used as the control.

2.10. X-Ray diffraction analysis

X-ray patterns of the samples were recorded using an X-ray diffractometer (XRD, D8 Advance, Bruker Inc., Germany). The diffractometer was worked at 40 kV and 40 mA. The diffractometer of scanning range was 5-40° for diffraction angle (2θ), and a scanning rate of 6°/min. The relative crystallinity (RC) was the percentage of the area of the crystalline region to the sum of the area of the crystalline and the amorphous region. The RC was calculated using origin 9.1.

2.11. Fourier transforms infrared (FTIR) spectroscopy

The FTIR spectra of the starch samples were recorded on a spectrometer (Thermo Electron Corporation, USA) at room temperature. The dried starch sample (2 mg) was mixed with KBr (150 mg) and pressed into slices. The scanning wavelength of spectrometer was 4000–400 cm⁻¹ and the resolution of spectrometer was 4 cm⁻¹ with 16 scans.

2.12. In vitro digestibility

Determined according to the method of Jiang et al. with some modifications [15]. 50 mg of the sample was added to 2 mL of distilled water then taken a water bath at 37 °C for 5 min. 8 mL of the prepared enzyme solution (mixed with pancreatin 3 × 10³ USP, amylosidase 40 U and 0.2 M sodium acetate buffer solution) was added and shaken in a water bath (37 °C, 300 rpm /min). At 0, 5, 15, 20, 30, 60, 120, 180, and 240 min of the reaction, 0.1 mL of the reaction solution was taken respectively, and 0.9 mL of anhydrous ethanol was added to terminate the reaction. The glucose content in the reaction solution was determined by GOPOD method. The absorption of the samples was recorded at 510 nm. The values for the digested starch fractions are expressed as milligrams of glucose × 0.9. The values for rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) were calculated from the G20 and G120 values.

2.13. Statistical analysis

All the analyses were performed in triplicate and ANOVA by Duncan's test (p < 0.05) was used to analyze the experimental data. SPSS 22.0 was used for statistical analysis.

3. Results and discussion

3.1. Total starch, resistant starch and amylose content of oat starch samples

The contents of protein, fat, water and ash in oat starch prepared by dilute alkali method were 0.13%, 0.32%, 7.31% and 0.36%, respectively. The total starch, resistant starch, amylose and amylopectin contents of OS and its resistant starch are shown in Table 1. At present, the preparation of starch by dilute alkali method is a widely used method. Except for ORS-C, which was 81.57%, the total starch content of each sample group was higher than 83%. ORS-B and ORS-C had similar levels of amylose and amylopectin. Among them, the content of amylose was up to 80% and the content of amylopectin was lower, about 20%. The ratio of amylose/amylopectin in ORS-B and ORS-C was 4, which was much higher than that of OS and ORS-A. The amylose content of OS and ORS-A was lower, only 23.24% and 31.87%. It showed that the added pullulan enzyme had a good effect on hydrolysis of amylopectin. In addition, higher levels of amylose contributed to the formation of resistant starch. The resistant starch content of ORS prepared by autoclaving-retrogradation cycle, enzymatic hydrolysis and ultrasound combined enzymatic hydrolysis respectively reached 9.44%, 30.15% and 36.15%, which were much higher than 5.31% of oat native starch. It indicated that ultrasound combined enzymatic hydrolysis method could effectively increase the content of resistant starch in oat starch. At the same time, the content of resistant starch in ORS-C was higher than that of ORS-B, indicating that ultrasound helped to increase the content of resistant starch. Ultrasound could improve mass transfer efficiency and promote the diffusion of oat starch molecules to the active site of pullulanase. In contrast to earlier findings, it was higher than the content of arrowhead-derived resistant starch (26.78%) prepared by Liang et al. [13] using ultrasound-assisted enzymolysis method, and the content of pea resistant starch (35.26%) prepared by You et al. [16] using ultrasound-assisted gelatinization process.

Table 1.

The total starch, resistant starch, amylose and amylopectin content of starch samples.

	Total starch (%)	Resistant starch (%)	Amylose (%)	Amylopectin (%)	Amylose/ Amylopectin
OS	83.26 ± 1.48a	5.31 ± 0.13a	23.34 ± 0.64a	76.66 ± 0.64c	0.3 ± 0.01a
ORS-A	83.61 ± 0.62a	9.44 ± 1.57b	31.87 ± 1.23b	68.13 ± 1.23b	0.47 ± 0.03a
ORS-B	83.58 ± 0.19a	30.15 ± 1.99c	80.19 ± 0.57c	19.81 ± 0.57a	4.05 ± 0.14b
ORS-C	81.57 ± 0.42b	36.15 ± 1.45d	79.89 ± 0.98c	20.11 ± 0.98a	3.98 ± 0.24b

OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch. Means with different small letter superscripts within the same column are significantly different at p < 0.05.

As a debranching enzyme, pullulanase can cleave the α-1, 6 glycosidic bonds in amylopectin to produce a large amount of amylose. Amylose aggregates and arranges to form tightly packed aggregates, thus forming resistant starch that is resistant to digestion [17]. In this study, the amylose content of ORS-B and ORS-C reached 80%, and the amylose/amylopectin ratio was also much higher than that of OS and ORS-A, which proved this point of view. Compared with ORS-B, the resistant starch content of ORS-C was increased by 19.90%, indicating that ultrasound treatment may promote the cleavage of starch molecules, reduce the molecular weight of starch, and improve the activity of pullulanase. In general, ultrasound treatment with appropriate time for enzyme reaction system could improve the efficiency of the enzyme reaction. At the same time, it also acted on the mixed system of the enzyme and starch. After ultrasound treatment, more sites on the free starch fragment that reacted with enzymes were exposed, increasing the efficiency of enzymatic reactions and leading to an increase in resistant starch content [18]. As the content of resistant starch increased, it helped to enhance its digestion resistance and reduce its GI value.

3.2. Solubility and swelling power of oat starch samples

The solubility of starch reflects the ability of amylose molecules to escape from starch granules at a certain temperature. The solubility and swelling power are closely related to the degree of interaction between starch chains in the crystalline and amorphous regions of starch granules [19]. The solubility and swelling power of starch samples are shown in Table 2. The solubility and swelling power of each group of samples increased with the increase of temperature. Possibly due to the increase in temperature, the interaction between starch molecules weakened. The structure of the starch granules was thus destroyed and the short-chain molecules spilled out, resulting in an increase in the amount dissolved in water. At the same time, due to changes in the structure of starch particles, water molecules entering the inside of starch could interact with starch molecules [20]. These results were consistent with the results reported by Li et al. for the preparation of purple yam resistant starch by autoclaving and multi-enzyme hydrolysis [21]. In addition, the source of the starch sample, the ratio of amylose/amylopectin, the preparation process and the thermal stability were closely related to the degree of interaction between starch and water [22]. At different temperatures, the solubility of ORS-B and ORS-C were higher than OS and ORS-A, indicating that ultrasound combined enzymatic hydrolysis effectively increased the number of short-chain starch molecules and reduced the hydrogen bonds between molecular chains, thereby increasing the water solubility of resistant starch. And the solubility and swelling power of all starch samples at 90 °C were significantly higher than those at 30 °C and 50 °C. This suggested that high temperatures could change the structure of the starch molecule. High temperature treatment converted the crystalline state of starch molecules into amorphous forms, and promoted water molecules to enter the amorphous region of starch particles, thereby affecting their solubility and swelling power [23].

Table 2.

The solubility and swelling power of starch samples.

	OS	ORS-A	ORS-B	ORS-C
Solubility (%)
30 °C	0.18 ± 0.03a	1 ± 0.07b	10.86 ± 0.03d	10.66 ± 0.7c
50 °C	0.28 ± 0.03a	1.32 ± 0.11b	11.97 ± 0.29d	11.23 ± 0.64c
90 °C	1.76 ± 0.24a	3 ± 0.42b	15.76 ± 0.17d	15.3 ± 0.18c
Swelling power (g/g)
30 °C	1.77 ± 0.06a	4.85 ± 0.06d	2.66 ± 0.03b	2.76 ± 0.02c
50 °C	1.83 ± 0.02a	5.31 ± 0.13d	2.81 ± 0.05b	2.88 ± 0.03c
90 °C	7.95 ± 0.18c	8.17 ± 0.43d	3.06 ± 0.06a	3.28 ± 0.01b

OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch. Means with different small letter superscripts within the same line are significantly different at p < 0.05.

However, the swelling power of ORS-A was the highest, indicating that the autoclaving-retrogradation cycle process helped to destroy the crystalline structure of starch molecules, thereby increasing the swelling power of resistant starch. Therefore, the solubility and swelling power of resistant starch prepared by different methods were significantly different.

3.3. Transparency and water holding capacity of oat starch samples

The transparency is one of the important external characteristics of starch paste, which has a great influence on the appearance and use of starch products. The transparency of starch samples is related to its source, amylose content, and molecular structure and so on. The transparency results of each sample are shown in Fig. 1A, which are ORS-B (38.61%), ORS-C (33.99%), ORS-A (26.98%) and OS (17.85%) in sequence. The reason for this result may be that the enzymatic hydrolysis destroyed the crystalline region and surface structure of starch, and the association between starch molecules was reduced, producing large amounts of amylose [24]. These leaded to an increase of starch solubility, an enhanced association between starch and water, and finally an increase in the transparency of starch granules [25]. These results were mutually confirmed with the determination results of amylose content and solubility of each starch sample.

Fig. 1.

(A) Transparency of starch samples; (B) Water holding capacity of starch samples. (OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch.) Different letters on the column chart indicate significant differences between groups (p < 0.05).

The water holding capacity reflects the ability of starch molecules to combine with water, which has a great impact on the processing of starch. It is related to the granule shape, molecular structure and amylose content of starch. The water holding capacity results of each sample are shown in Fig. 1B, and the order from high to low is ORS-A (714.97%), ORS-C (378.26%), ORS-B (334.23%), OS (188.65%). These data results differed from the swelling power of each starch sample at 30 °C, but the effects were proportional. This indicated that the destruction of starch granule structure caused by the autoclaving-retrogradation cycle process could effectively improve the water holding capacity of starch. At the same time, the results of water holding capacity were similar to arrowhead-derived resistant starch prepared by Liang et al. by traditional enzymolysis and ultrasound-assisted enzymolysis method [13]. In addition, the debranching process produced a large amount of amylose, which increased the number of hydrophilic hydroxyl groups [20]. The water holding capacity of ORS-C was higher than that of ORS-B, indicating that ultrasound also helped to reduce the chain length of amylose and increase the number of hydrogen bonds. Amylose typically formed chain aggregates through hydrogen bonding and hydrophobic interactions, thereby improving the water holding capacity of starch [14].

3.4. Morphological properties of oat starch samples

The morphological characteristics of each group starch samples were studied by scanning electron microscopy, as shown in Fig. 2. OS had particles of different sizes, with an average particle size of about 18 μm. It was mainly oval or polygonal with smooth surface, which was the same as the morphological characteristics of oat starch reported by Shah et al. [12]. The shape of ORS-A was significantly different from that of OS. The particle size distribution of ORS-A was larger, with an average particle size of 148 μm, in the form of irregular fragments and increased surface roughness. The changes in the morphological characteristics of ORS-A could be due to the autoclaving-retrogradation cycle process that leaded to the rupture of starch granules and the formation of irregular granule aggregates. And this special shape of irregular aggregates had a certain resistance to digestion [26]. The average particle size of oat resistant starch ORS-B and ORS-C were similar, and they also had larger size distribution, but both were smaller than ORS-A. ORS-B and ORS-C were irregular in shape, the surface of ORS-C had more groove-like strips, which may be caused by the ultrasound treatment applied during the enzymatic hydrolysis of ORS-C. When cavitation bubbles were broken due to ultrasound, a high-pressure gradient and local velocity were created near the bubbles, creating shear forces, damaging starch particles and cutting long chains. The cavitation effect of ultrasound acted on the enzymatic hydrolysis system to destroy the surface shape of starch granules, generate some cracks and further promote enzymatic hydrolysis. Therefore, there were differences in the morphological characteristics of the formed RS and affected the resistant starch content [27]. This was also one of the main reasons why the resistant starch content of ORS-C prepared by ultrasound combined enzymatic hydrolysis was higher than ORS-B prepared by traditional enzymatic hydrolysis. Therefore, compared to ORS-B, the digestibility of ORS-C would be reduced, and the GI value would also be reduced. In addition, the special structure of ORS-C could be one of the factors that contribute to its production of short-chain fatty acids in the large intestine as a substrate for intestinal microbial fermentation [28].

Fig. 2.

SEM micrographs of starch samples (magnification: 200 × and 2000 × ). (OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch.).

3.5. Particle size distribution of oat starch samples

The particle size distributions of oat starch and resistant starch are shown in Fig. 3A and Table 3. The volume average particle size D [3,4], surface area average particle size D [2,3] and Dv (10), Dv (50), Dv (90), span of oat resistant starch in each group were significantly higher than those of oat starch, and the results were consistent with corn resistant starch prepared by Li at el. by an autoclaving and autoclaving-microwave method [29]. Among them, D [3,4] (164.59 μm), D [2,3] (44.76 μm), Dv (50) (148.40 μm), Dv (90) (322.54 μm) and span (2.03) of ORS-A were all the largest. This indicated that during the autoclaving-retrogradation cycle process, the autoclaving gelatinization caused the starch granules to be damaged and swelled. Then during the retrogradation process, starch molecules re-orderly combined to form crystals, which eventually leaded to a significant increase in the starch particle size and span. The aggregates formed by the rearrangement and combination of amylose molecules produced by enzymatic hydrolysis and ultrasound combined enzymatic hydrolysis also increased the particle size of starch granules, but not as much as the autoclaving-retrogradation cycle. The span of ORS-C was significantly lower than that of ORS-B, indicating that ultrasound treatment could promote uniform distribution of starch particles and increase dimensional consistency. In addition, there was no significant difference in particle size between ORS-B and ORS-C, indicating that ultrasound did not significantly increase the particle size of ORS-C. This differed from the findings of Ding et al. and may be related to the time and power of the ultrasound [30]. When the ultrasound time was short or the power was small, the particle size change of resistant starch was not significant. At the same time, a number of studies have reported that resistant starch with different sizes had different physicochemical properties and functional properties [31]. For example, the size of the surface area of starch granules would affect the digestion of enzymes [14]. Due to the different particle sizes of resistant starch granules, the surface area of enzyme action was also different, which directly affected the digestion of enzymes.

Fig. 3.

(A) The particle size distributions of starch samples; (B) XRD diffractograms of starch samples; (C) FTIR spectra of starch samples. (OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch.).

Table 3.

Diameters of starch samples.

	D[2,3] (μm)	D[3,4] (μm)	Dv(10) (μm)	Dv(50) (μm)	Dv(90) (μm)	Span
OS	9.37 ± 0.18a	18.32 ± 0.01a	10.05 ± 0.01a	18.14 ± 0.02a	27.94 ± 0.02a	0.98 ± 0.00a
ORS-A	44.76 ± 0.16c	164.59 ± 0.17c	21.83 ± 0.09b	148.4 ± 0.54c	322.54 ± 0.56c	2.03 ± 0.01d
ORS-B	36.09 ± 0.64b	128.56 ± 0.31b	27.79 ± 0.11c	116.78 ± 0.2b	246.25 ± 0.73b	1.87 ± 0.01c
ORS-C	38.82 ± 0.11b	124.85 ± 0.15b	31.32 ± 0.04d	112.53 ± 0.28b	237.21 ± 0.03b	1.82 ± 0.01b

OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch. Means with different small letter superscripts within the same column are significantly different at p < 0.05.

3.6. Thermal properties of oat starch samples

The gelatinization onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), gelatinization temperature range (Tc-To) and gelatinization enthalpy (△H) of starch samples are shown in Table 4. The gelatinization onset temperature of oat starch OS was the highest, indicating that oat starch needed a higher gelatinization temperature to destroy its crystalline structure. Compared with OS, all the gelatinization indexes of oat resistant starch ORS-A were decrease, which was consistent with the research of Li et al. [32]. The decrease in gelatinization temperature of ORS-A may be due to the rearrangement of starch chains during retrogradation, which destroyed the crystalline structure of starch granules, resulting in a decrease in the stability of the double helix structure. It further caused the reduction of the gelatinization enthalpy and the gelatinization temperature of ORS-A. The gelatinization indexes of ORS-B and ORS-C had no significant difference, but other gelatinization indexes were significantly higher than OS except for the onset temperature of gelatinization. This was consistent with the results of Flores-Silva et al. on the ultrasound-treated of corn starch, which had less effect on the gelatinization temperature of starch granules [33]. Studies reported that the gelatinization temperature and crystallinity of starch were closely related to the amylose content and double helix structure in starch [34]. ORS-B and ORS-C produced a large amount of amylose by enzymatic hydrolysis, then formed a large number of new double helix structures at low temperature. And the structure of the polymer was tighter and the crystallinity and number of crystals were higher. Tc-To of starch reflects the stability and uniformity of microcrystals in starch, and a larger gelatinization temperature range indicates that there are more microcrystals in the crystalline region [35]. The gelatinization enthalpy △H reflects the melting of the double helix structure. The number of double helix structures in ORS-B and ORS-C increased, and the melting of double helix structures required more energy, so its enthalpy value also increased accordingly. Higher enthalpy values indicated a higher number of double helixes, tighter packing and better thermal stability. The high crystallinity of ORS-B and ORS-C also confirmed this result. However, Shah et al. modified oat starch by double enzymatic hydrolysis had a low △H value (11.06 J/g). It could be that the continuous enzymatic digestion of the two enzymes led to an increase in the proportion of short amylopectin chains and a decrease in the length of amylopectin chains, resulting in a decrease in the number of double helixes, so the energy required to melt the crystal structure was low [13]. Therefore, the thermal stability of oat resistant starch prepared by ultrasound combined enzymatic hydrolysis was higher than that of dual enzyme modified oat starches.

Table 4.

The thermal properties of starch samples.

	To(°C)	Tp(°C)	Tc(°C)	Tc-To(°C)	△H(J/g)
OS	56.98 ± 0.06c	67.87 ± 0.33b	79.1 ± 0.51b	22.12 ± 0.45a	11.05 ± 0.42b
ORS-A	45.5 ± 0.45a	57.91 ± 0.33a	66.76 ± 0.01a	21.26 ± 0.46a	1.88 ± 0.01a
ORS-B	49.74 ± 1.17b	76.78 ± 0.03c	110.55 ± 0.54c	60.81 ± 1.71b	16.43 ± 0.49c
ORS-C	49.52 ± 0.74b	76.05 ± 0.05c	108.84 ± 0.54c	59.33 ± 0.21b	15.28 ± 0.47c

OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch. Means with different small letter superscripts within the same column are significantly different at p < 0.05.

3.7. Crystalline characteristics of oat starch samples

The crystalline type and relative crystallinity of starch samples can be determined and analyzed by x-ray diffractometer, and its crystalline types can be divided into A-type (monoclinic unit cell), B-type (hexagonal unit cell), C-type (mixture of A-type and B-type) and V-type (mostly in starch lipid complexes and gelatinized starch). The crystalline structure of resistant starch is related to its origin and processing method. An example of this is A-type starch could be changed into B-type, V-type or B + V-type resistant starch after different processing methods [19]. The X-ray diffraction patterns and relative crystallinity of the starch samples in this study are shown in Fig. 3B and Table 6, respectively. Oat starch OS had a single peak at 2θ angles of 15° and 23°, and double peaks at 17° and 18°. It was a typical A-type structure and the relative crystallinity of OS was 45.03%. After the autoclaving-retrogradation cycle, ORS-A appeared diffraction peaks at 2θ angles of 5.6°, 17°, 19.8°, and 22°, the crystalline type changed to B + V-type, and the relative crystallinity increased to 53.79%. This result was consistent with the research of Ratnaningsih et al. [36], and also confirmed the previous view that the A-type structure could change to B + V-type structure after gelatinization.

After enzymatic hydrolysis and ultrasound combined enzymatic hydrolysis, ORS-B and ORS-C appeared diffraction peaks at 2θ angles of 5.6°, 15.2°, 17.1°, 22°, and 24°, respectively, the crystalline types changed to B + C-type. The relative crystallinity of ORS-B was 64.37% and the relative crystallinity of ORS-C increased to 66.98%. This result of relative crystallinity was higher than arrowhead-derived resistant starch (55.6%) prepared by Liang et al. [13]. The relative crystallinity of ORS-C was the highest, indicating that the ultrasound combined enzymatic hydrolysis process could promote the rearrangement of amylose in the crystalline and non-crystalline regions to form a more stable crystalline structure and improve the relative crystallinity [37]. The shear force generated by ultrasound cavitation increased the free fragments in starch aggregates, as well as the enzyme contact site, which helped enzymatic hydrolysis to produce a large amount of amylose. The increased amylose reassembled into a double helix through dense interactions, forming tight crystals [38]. In addition, the crystalline region of starch was not easily degraded by enzymes. The relative crystallinity of ORS-C was the highest and the crystallization region was the largest, indicating its strong resistance to digestion. Shen et al. prepared ORS by enzymatic hydrolysis and autoclaving- retrogradation, with large average particle size, irregular surface, B-type crystals. The GI value of ORS was 48, which was lower than that of oat native starch [39]. It was shown that oat resistant starch ORS-C prepared by ultrasound combined enzymatic hydrolysis could also be used as a raw material for the preparation of low GI foods.

3.8. FTIR spectra of oat starch samples

FTIR spectroscopy can study the molecular structure and short-range order of resistant starch by detecting stretching, bending, and deformation corresponding to the main functional group features [40]. As shown in Fig. 3C, the spectra of the starch samples were similar and no new absorption peaks appeared. It was shown that the resistant starch prepared by different methods neither produced new chemical groups. This proved that autoclaving-retrogradation cycle, enzymatic hydrolysis and ultrasound combined enzymatic hydrolysis belonged to the physical modification process [41]. The absorption peaks of samples were displayed at 3432 cm⁻¹ (–OH stretching vibration), 2927 cm⁻¹ (deformation of –CH₂ and O-H groups), 1645 cm⁻¹ (C = O stretching vibration), 1156 cm⁻¹ (C-O with some C-O-H contributions stretching vibration) and 1078 cm⁻¹ (C-O of glycosidic bond stretching vibration) [42]. The spectral absorption intensity of oat starch at 3432 cm⁻¹ was the lowest, indicating that the number of hydrogen bonds generated by amylose increased due to the free hydroxyl groups, intermolecular and intramolecular stretching vibrations in the resistant starch prepared by different methods. The peak absorption intensity of ORS-A at 2927 cm⁻¹ was the largest, indicating that the vibrational conformational changes of the O-H and –CH₂ groups related to retro genesis were larger. The peak at 1645 cm⁻¹ reflected the water absorption capacity of the amorphous part of starch. The peak intensity of ORS-A was also the highest, which was consistent with the strongest water holding capacity of ORS-A. In addition, the absorption intensities of starch samples at 1156 cm⁻¹ and 1078 cm⁻¹ were also different. Studies showed that the peak intensity at 1022 cm⁻¹ was related to the vibrational mode of the disordered or amorphous part of the starch molecule, and the peak intensity was inversely proportional to the crystallinity. The peak intensity at 1047 cm⁻¹ was related to the high crystallinity and ordered structure of starch molecules. The peak intensity ratio of 1047/1022 cm⁻¹ (R_1047/1022) was used to represent the changes in the crystallinity and ordered structure of starch molecules [43]. The R_1047/1022 of OS, ORS-A, ORS-B and ORS-C were 0.889, 0.898, 0.955 and 0.998, respectively. The R_1047/1022 of ORS-C was the largest, indicating that ORS-C had the highest crystallinity (consistent with the crystallinity results in Table 5), the most ordered molecular structure and the shortest molecular ordered structure. It may be that ultrasound combined enzymatic hydrolysis enhanced the binding between amylose chains and promoted the formation of relatively ordered structure of starch molecules. The above results indicated that the crystallinity and conformational properties of starch molecules changed during the processing of native starch, and these properties were also affected by different processing methods. Due to the high crystallinity and molecular structure order of ORS-C, its digestion resistance was also the strongest. These results would been verified in the in vitro digestibility determination of oat starch samples.

Table 5.

The relative crystallinity, crystalline type, RDS, SDS and RS contents of starch samples.

	Relative crys tallinity (%)	Crystalline type	RDS (%)	SDS (%)	RS (%)
OS	45.03 ± 0.58a	A	57.33 ± 2.71c	36.09 ± 4.91bc	6.58 ± 2.20a
ORS-A	53.79 ± 0.19b	B + V	44.54 ± 1.70b	42.13 ± 0.73c	13.34 ± 0.97b
ORS-B	64.37 ± 0.54c	B + C	38.9 ± 1.81a	29.20 ± 0.57ab	31.90 ± 2.39c
ORS-C	66.98 ± 0.35c	B + C	38.52 ± 0.56a	24.67 ± 0.36a	36.81 ± 0.92d

OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch. RDS, SDS and RS represent rapid digestible starch, slow digestible starch and resistant starch respectively. Means with different small letter superscripts within the same column are significantly different at p < 0.05.

3.9. In vitro digestibility of oat starch samples

The response to postprandial blood glucose depended largely on the amount of rapidly digestible starch (RDS). Higher resistant starch (RS) and slow digestible starch (SDS) levels in starch helped control elevated glycemic index (GI). Due to ORS-C had the highest content of resistant starch, its differences with other oat starch samples in vitro digestion characteristics were further investigated. The oat starch was gelatinized and dried before performing in vitro digestion experiments. As shown in Fig. 4A and 4B, the digestible hydrolysis rates of ORS-A, ORS-B and ORS-C were significantly lower than that of gelatinized oat starch OS. Among them, ORS-C had the lowest digestible hydrolysis rate, indicating that it had the strongest digestion resistance ability. This also verified the result of the highest resistant starch content of ORS-C. As shown in Table 5, the contents of RDS, SDS and RS in gelatinized oat starch OS were 57.33%, 36.09% and 6.58%, respectively. The SDS and RS contents of ORS-A, ORS-B and ORS-C were significantly increased compared to OS. The RS contents of oat starch samples were slightly higher than that measured directly by the RS kit, which could be due to the fact that different detection methods also had some differences in the determination results of the samples. The RDS, SDS and RS contents of ORS-C were 38.52%, 24.67% and 36.81%, respectively. This indicated that compared with other oat starch samples, the RS content of ORS-C prepared by ultrasound combined enzymatic hydrolysis was significantly increased, the digestion rate was reduced and the resistance to digestion was improved. This was closely related to the special structure and higher amylose content of ORS-C [44]. ORS-C had the strongest digestion resistance because of its roughest surface morphology, highest crystallinity, most ordered molecular structure, larger average particle size and B + C-type crystallization. Fig. 4C was a heat map of the correlation between hydrolysis rate and other indicators of oat starch samples. It could be seen from the figure that the hydrolysis rate was strongly negatively correlated with RS content, amylose content, relative crystallinity and R_1047/1022, and weakly negative with average particle size. When the hydrolysis rate of starch decreased, its anti-digestion properties were enhanced. Therefore, the anti-digestion properties of starches were strongly positively correlated with RS content, amylose content, relative crystallinity and R_1047/1022, and weakly positively correlated with average particle size. That is, when ORS had higher RS content, amylose content, relative crystallinity and R_1047/1022, its digestion resistance was stronger. In addition, the content of indigestible starch (SDS + RS) in ORS-C reached 61.48%, which could be used as a material for embedding drugs and had a slow-release effect. It could also be used to prepare resistant starch products with physiological effects such as weight loss [45], lowering blood sugar [46], relieving symptoms of constipation [47] and slowing down the progression of chronic kidney disease [48] etc. Due to the strong anti-digestion properties of ORS-C, it was especially suitable for making foods with low glycemic index for diabetics.

Fig. 4.

(A) The hydrolysis rate of starch samples; (B) The hydrolysis rate of starch samples at 240 min; (C) Heat map of hydrolysis rate correlation with other indicators. (OS, oat starch; ORS-A, autoclaving-retrogradation cycle treated oat starch; ORS-B, enzymatic hydrolysis oat starch; ORS-C, ultrasound combined enzymatic hydrolysis oat starch.).

4. Conclusions

In the present study, oat resistant starch was prepared by autoclaving-retrogradation cycle, enzymatic hydrolysis and ultrasound combined enzymatic hydrolysis. Their physicochemical properties, structural characteristics and in vitro digestion characteristics were studied. Among them, ORS-C prepared by ultrasound combined enzymatic hydrolysis had a B + C-type crystal structure, with the highest content of resistant starch, larger particle size, the smallest span value, the highest relative crystallinity, the most ordered and stable double helix structure and the roughest surface irregular shape. These structural features were closely related to the preparation method of resistant starch. The anti-digestion properties of ORS-C was strongly positively correlated with RS content, amylose content, relative crystallinity and R_1047/1022, and weakly positively correlated with average particle size. At the same time, the solubility, swelling power, transparency, water holding capacity and thermal stability of ORS-C were also significantly improved, and it had the best anti-digestion properties. These properties were closely related to the structural characteristics of resistant starch. According to the strong digestion resistance of oat resistant starch prepared by ultrasound combined enzymatic hydrolysis, ORS-C could be widely used in development of some functional foods with low GI and improved processing properties.

CRedit authorship contribution statement

Ji’an Xia and Yu Zhang contributed equally to this work. Conception and design of the research: Yu Zhang, Qiqi Sun, Man Wang, Zhenliang Sun, Suhua Zhang and Xiao Guan; performing experiments, analysis of data and preparation of figures: Ji’an Xia; interpretation of the results of experiments, drafting the manuscript: Ji’an Xia and Yu Zhang; and funding: Kai Huang and Hongwei Cao.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.