Starch isolated from different hulless barley cultivars differs in their chemical and structural characteristics

Abstract This study aimed to isolate starch and evaluate its chemical and structural characteristics from six Chinese hulless barley (HB) cultivars. Starch isolated from naked barley displays A‐type crystalline packing and a regular granular shape. We measured peak viscosity values ranging from 237 to 264 cP, trough viscosity values from 305 to 380 cP, breakdown values from 390 to 535 cP, final viscosities from 357 to 523 cP, setback values from 245 to 354 cP and 383 to 460 cP, peak times from 5.53 to 5.73 min, and pasting temperatures from 93.10 to 94.65°C by RVA. Transition temperatures (T 0, T p, and T c), gelatinization temperature ranges (ΔT r), and enthalpies of gelatinization (ΔH) were measured on a differential scanning calorimeter analyzer (DSC) and ranged from 57.81 to 61.25°C, 64.36 to 67.57°C, 82.03 to 84.70°C, and 21.52 to 26.89°C and 7.14 to 10.66 J/g, respectively. The varying chemical and structural characteristics of HB starch isolated from different cultivars suggested the potential for broader applications of the cereal.


2011)
, where it has been a foodstuff in Tibet since the 5th century.
HB has the nutritive value of wheat and hulled barley, can be metabolized, and has a high protein and low fiber content (Rezaei, Dehghan, & Ayatollahy, 2008). HB is used in animal feed in areas of reduced rainfall but has attracted interest among researchers because of its solubility, high malt quality, ease of preparation, and β-glucan and arabinoxylan content Lin et al., 2018;Wang et al., 2011).
Further knowledge of the starch characteristics is therefore required.
To date, the structural and chemical properties of HB starch have not been compared in different Chinese samples. Here, we studied the morphology, heat, and gelatinization characteristics of six HB starch samples isolated from major producing areas in China. X-ray powder diffraction (XRD) and scanning electron microscopy (SEM) were used to study particle characteristics, paste properties were measured by RVA, and thermal properties were evaluated by differential scanning calorimeter (DSC). The currently selected variety is a newly developed variety that has a large cultivation volume, high yield, and strong adaptability in Tibet in the past 10 years, and basically covers the main varieties of the current barley in Tibet. Among them, black barley is a newly developed variety containing anthocyanins in Tibet, and the others are white barley. It is worth noting that the starch of the new main cultivar has not been studied and compared, and it has research value, and it is easy to collect in Tibet, which is conducive to experimentation. Moreover, the differences in the nature of these six species are also obvious. The results of this study have great guiding significance for the deep processing of barley. After studying and comparing their properties, they can have a clear understanding of the advantages and disadvantages of each species and use their unique advantages to apply to their suitable fields.  Li et al., 2014). Briefly, grains were washed in water and then immersed in 0.1% anhydrous sodium sulfite (solution/grain ratio = 6:1) at 20°C for 48 hr while stirring. After removing the soaking liquid, the grains were washed in water. Samples were homogenized in a pulping machine, following which the slurry was passed through mesh nylon. Residues were washed in water to release all the starch, and remnants on the cloth were discarded. Samples were centrifuged at 2504 g for 12 min, and the supernatants were discarded. The upper precipitate (pigment layer) was removed, and the starch was separated by resuspending in water. Samples were centrifuged, the starch paste was washed in ethanol (95%), and samples were centrifuged three times. Supernatants were discarded, and the starch was oven-dried at 50°C for 12 hr.

| Starch granule morphology
Morphology was assessed by scanning electron microscope (SEM; Li et al., 2014). Starch samples were prepared in aluminum sample holders sealed with carbon tape metalized with gold to promote the reflection of electron beams. The samples were imaged on a ZEISS EVO 18 SEM (under a vacuum of 1.5 × 10 −3 Pa, magnification = 500 and 2,000×, acceleration voltage = 3.0 kV).

| Granule size
The sample was stirred in ethanol at 626 g (Mohapatra et al., 2018), and then, the particle size was measured using a laser diffraction particle size analyzer (Mastersizer 2000).

| Amylose content
The amount of amylose in various starch was measured using colorimetric assays described by Zavareze group (Bruni, Oliveira, Halal, & Flores, 2017). Briefly, 100 mg of the samples was added to 100-ml flasks and mixed with 96° GL ethanol and 1 mol/L NaOH. The samples were incubated at 100°C for 10 min in a water bath and cooled for 30 min, and distilled water was added. Aliquots (5 ml) were transferred to 100-ml flasks containing acetic acid (1 ml, 1 mol/L) and iodine (2 ml of 2% [w/v]) and filled with water. Data were calculated based on the standard curves of pure amylose (Sigma). The absorbance was measured at 610 nm.

| Crystallinity
The crystallinity of the starch was analyzed by obtaining an Xray diffraction pattern on a D8 Advance XRD (Bruker AXS). The crystallinity was evaluated on MDI Jade 6 software to fit the measured XRD peak. R% crystallinity is used to measure the ratio of peak area, background area, and peak area (Liu et al., 2017).

| Viscosity
In a typical procedure, 2.0 g of starch is suspended in 28 g of water, incubated at 30°C for 2 min, incubated at 95°C for 8 min, and cooled at 50°C for 8 min. Adhesive properties were evaluated in the literature using RVA-3C (Newport Scientific; Blazek & Copeland, 2008).

| Thermal properties
Thermal analysis of the starch was performed according to the method described by the Zhu. F team (Zhu & Xie, 2018). The enthalpy and peak gelation based on the starting temperature were measured. Two milligram of samples was weighed in an aluminum capsule, and 7 μl of water was added; then, the sealed capsule was allowed to stand for 30 min, followed by a PerkinElmer DSC 8500 at 20-120°C/min flow rate for thermal analysis (under a nitrogen atmosphere; scanning rate of 50 ml/min; Zhu & Xie, 2018).

| Statistical analysis
The data obtained were the average values of the six HB varieties (n = 3). A two-way ANOVA test was used for statistical analysis using ANOVA and Tukey's test. Analysis was performed by SPSS 16.0 statistical software. p < 0.05 indicates that the difference is statistically significant.

| Characterization of starch granule
From the SEM results in Figure 1, it can be observed that the starch granules are oval, spherical, and polygonal. Moreover, the particle size of the particles ranges from 10 to 20 μm. It can be seen from Table 1 that the average particle diameter of the starch is from 18.99 to 23.17 μm. Among them, the starch having the smallest particle diameter is Dongqing 11, and the average particle diameter is 18.99 μm. The larger particle sizes are Nakano blue 25, Black HB, and Dongqing 18, and the average particle diameters are 20.33, 22.51, and 23.17 μm, respectively. In particular, it was found from the comparison of the overall morphology that the starch of the Black HB variety showed a state of particle agglomeration. On the contrary, the holly 18 was scattered and evenly distributed, and it was not easy to agglomerate. It is speculated that this result is related to the viscosity of the starch.

| Evaluation of amylose
As can be seen in Table 1, the amylose content of the six HB varieties was 12.71%-15.72%. Among them, Zangqing 2000 has the highest amylose content, while Black HB has the lowest content. It has been reported that extensive variation in amylose is influenced by the type of variety and method of determination. The ratio of amylose affects starch paste viscosity and amylose content affects functional/physicochemical properties due to degradation (Blazek & Copeland, 2008;Kossmann & Lloyd, 2000;Uarrota et al., 2013). It is worth noting that many human nutrition research institutes abroad have done many experiments to confirm the healthcare value of amylose. High-amylose foods are ideal for diabetics. High-amylose starch is also an ideal food for patients with gallstones and hypertension and has the effect of preventing gallstone formation and lowering blood cholesterol. Amylose has stronger tensile strength and good formability, which can increase the brittleness and strength of the product. Therefore, Zangqing 2000 has a high amylose content, which may play a huge application value in the field of food production, processing, and even packaging materials.

| Crystallinity assessment
As can be seen from Figure 2, the six starch samples showed a similar XRD pattern at 15° and 23° 2θ, and a doublet of 17° and 18° 2θ, with the type A crystallinity observed in the grain starch. The peak positions are consistent. Subsequently, the crystallinity of different starches is also summarized in Table 2. It can be found that Dongqing 18 starch has the highest crystallinity (31.06%) and Black HB has the lowest (11.81%). In particular, comparing the amylose content, it was found that there was no significant correlation between crystallinity TA B L E 1 Granule size and amylose % of starch obtained from HB cultivars  Note: All values are the means of triplicate determinations ± SD. The means within columns with different letters are significantly different (p < 0.05). b: d (0.1), d (0.5), and d (0.9) = granule sizes at which 10, 50, and 90% of granule volume are smaller, respectively; D (3,2) = surface-area-weighted mean diameter; D (4,3) = volume-weighted mean diameter.
F I G U R E 2 X-ray diffraction spectra of HB starch (A); RVA profile of the isolated starch granules (B); DSC thermographs of the starch samples (C)

| Pasting characteristics
Characteristics of the starch samples are shown in Table 3, and the pasting curve is shown in Figure 2.  (Bhatty, 1997;Lopez-Rubio et al., 2008). Breakdown viscosity is the difference between peak and trough viscosity and represents the degree of granule disintegration, swollen granule disruption, and amylose release during breakdown. Dongqing 18 had the minimum peak viscosity and breakdown values suggestive of strong cohesive force within its starch granules, thermal stability, and low levels of degradation (Kaur, Singh, Ezekiel, & Guraya, 2007).
Importantly, the final viscosity is a measure of the stability of cooked paste, which tends to increase with cooling owing to the accumulation of resident amylose. The setback viscosity is a measure of the final viscosity and the peak viscosity, the difference being the measure of the viscosity of the heated starch paste and the rate of degradation after gelatiniza-  Figure 2 shows the DSC thermographs of the HB starch samples.

| DSC
The onset gelatinization temperature (T 0 ), conclusion temperature (T c ), peak temperature (T p ), gelatinization temperature range (ΔT r ), and enthalpies of gelatinization (ΔH) significantly differed among samples. The transition temperature (T 0 , T p , and T c ), ΔT r ,  (Kaur et al., 2009;Miao, Zhang, & Jiang, 2009). However, further studies are required to assess the desirable characteristics and commercial application of each starch through the analysis of their structural, physical, and functional properties.

| CON CLUS ION
Studies investigating the impact of modifications on the structural and physiochemical properties of HB starch sampled from the different cultivars are also warranted.  (201810693032).

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.