Combined ultrasound and germination treatment on the fine structure of highland barley starch

Graphical abstract

Highlights

• •On the highland barley starch, the impacts of ultrasound treatment and germination were examined.
• •U-G treatment resulted in higher Mws in samples than G-U treatment.
• •The polymerization degree of the branched starch chain lengths was slightly changed.
• •With increasing germination time, all test groups demonstrated a reduction in particle size distribution range.
• •Ultrasound pretreatment followed by germination improves the intricate morphology of highland barley starch.

Abstract

Highland barley is a grain crop grown in Tibet, China. This study investigated the structure of highland barley starch using ultrasound (40 kHz, 40 min, 165.5 W) and germination treatments (30℃ with 80% relative humidity). The macroscopic morphology and the barley's fine and molecular structure were evaluated. After sequential ultrasound pretreatment and germination, a significant difference in moisture content and surface roughness was noted between highland barley and the other groups. All test groups showed an increased particle size distribution range with increasing germination time. FTIR results also indicated that after sequential ultrasound pretreatment and germination, the absorption intensity of the intramolecular hydroxyl (–OH) group of starch increased, and hydrogen bonding was stronger compared to the untreated germinated sample. In addition, XRD analysis revealed that starch crystallinity increased following sequential ultrasound treatment and germination, but a-type of crystallinity remained after sonication. Further, the Mw of sequential ultrasound pretreatment and germination at any time is higher than that of sequential germination and ultrasound. As a result of sequential ultrasound pretreatment and germination, changes in the content of chain length of barley starch were consistent with germination alone. At the same time, the average degree of polymerisation (DP) fluctuated slightly. Lastly, the starch was modified during the sonication process, either prior to or following sonication. Pretreatment with ultrasound illustrated a more profound effect on barley starch than sequential germination and ultrasound treatment. In conclusion, these results indicate that sequential ultrasound pretreatment and germination improve the fine structure of highland barley starch.

1. Introduction

It is well known that starch is a primary source of energy that can be derived from the consumption of staple foods, such as cereals. Despite this, there is an increasing level of concern among consumers regarding the effects of refined starch consumption on their health. Developing new starch resources and methods for improving current starches is very important. Presently, researchers are focusing on developing new starches through the investigation of the properties of barley, potato, pea, and corn, along with other grains and beans, as well as the modification of various properties of traditional starches. For example, Xie et al. [1] assessed the effect of various drying techniques on the fine structure of wheat (A-type), potato (B-type), and pea (C-type) starches. They concluded that freeze-drying reduced their relative crystallinity and disrupted the ordered structure of B-type starch. In contrast, oven drying did not significantly alter the structure of starch. It has been reported that the calcium and magnesium-fortified potato starches have reduced their peak viscosity and disintegration values [2]. In contrast, their pasting temperature and enthalpy are similar to those of the original starch. Waleed et al. [3] found that fermentation did not affect the fine structure of highland barley starch, but the spacing of pores and the degree of surface breaking were increased. Liang et al. [4] demonstrated that ultrasound could induce germination of maise seed, increase the growth rate of maise plumule, and increase the content of reducing sugars, soluble proteins, and γ-aminobutyric acid.

With frequencies above 20 kHz, ultrasound can enhance adsorption with minimal energy consumption and increase yields [5], [6], [7]. It can enhance seed germination through biological, chemical and physical methods. Ultrasound affects germination in three ways: (1) Mechanical effects: mechanical energy is transmitted with ultrasound. Barley seeds can be broken through ultrasonic treatment of the shell; ultrasonic pretreatment increases rice grains' surface pores and cracks, enhancing the hydration process. (2) Cavitation effect: Due to the sparse and dense nature of ultrasound waves, these waves can cause rapid contractions and expansions of plant cells, resulting in the formation of bubbles. It has been reported that the cavitation effect of ultrasound can destroy the cell wall, allowing water molecules to enter the cell, thus accelerating the rehydration rate easily. (3) Thermal effect: the acoustic energy of the ultrasound is converted into thermal energy within the medium as a result of mutual friction, which warms the medium. Cavitation effect can also be described as an instantaneous thermal effect. An earlier study of sequential germination and ultrasound treatment (Table 1) revealed that ultrasound exhibited a more significant effect on germination. In addition, the structure of grain starch also changed under the influence of ultrasound. Wang Ning et al. [8] found that in pea starch extracted with ultrasound-assisted alkali, ultrasound not only affected the morphological characteristics of starch particles, but also made the starch have higher amylose content, water solubility, swelling power and viscosity.

Table 1.

Recent advances in ultrasound-assisted germination of plant seeds.

Seed	Ultrasonication conditions	Ultrasonic improvements	References
Maise	30 ℃, 45 kHz, 30 min	Sugar content increased by 22.83%, soluble protein content increased by 22.52%, γ -GABA content increased by 30.55%, glutamate decarboxylase activity was higher than the control group, and the γ -amino butyrate enrichment time was shortened	Zhang et al. [19]
Brown rice	28 kHz, 400 W, time (5, 10, 15 and 30 min)	The germination process of brown rice was accelerated, with a significant reduction of reducing sugar content, particle size, and enhancement of γ -aminobutyric acid, antioxidants, and proline	Xia et al. [15]
Highland barley	Drying temperatures of 55 ℃ and 70 ℃, ultrasonic intensities of 125.1 W/dm2 and 180.2 W/dm2, and a drying mode of 5 s on/5 s off	Organic selenium preservation was improved by increasing drying speed, reducing drying time, and increasing the rehydration rate	Song et al. [18]
Brown rice; Red rice	23–24 ℃,25 kHz， 5 min and the acoustic power density (APD) was 16 W/L	Surface microstructure changes indicate enhanced starch hydrolysis, increased glucose content, decreased viscosity, and significantly decreased energy consumption during germination	Ding et al. [20]
Barley seed	30 ℃, 20 kHz, total electrical power of the device was 460 W, and time (5, 10 and 15 min)	Improved the α -amylase activity and shortened the germination period	Yaldagard et al. [21]
Barley seed	30 ℃，20 kHz, power input of 20, 60, and 100% of 460 W, time (5, 10 and 15 min)	Germination rates increased by about 1.042–1.065 times, and the germination period was shortened by 30–45%	Yaldagard et al. [22]

The Highland barley (Hordeum vulgare Linn Var. Nudum Hook. f.) is the main grain grown in Tibetan areas of China, and it is used in food, medicine, and health products [9]. Germination is a low-cost, low-energy, and non-polluting method of biomodifying grains. It has been demonstrated that germination can increase grains' antioxidant properties, reducing cholesterol and blood glucose levels [10], [11]. The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is also produced during the germination process of highland barley. Various studies have shown that it lowers blood pressure, induces relaxation, and enhances immune function during stress [12], [13], [14]. Although sequential ultrasound pretreatment and germination increase the GABA content of brown rice, red rice, and wheat grains [15], [16], [17], it is unknown whether the increased GABA concentration would impact the structure and properties of starch. Song et al. [18] examined the drying characteristics, microstructure and bioactivity characteristics of ultrasound-assisted hot-wind drying of germinated highland barley; however, the impact of ultrasound pretreatment on the properties of highland barley starch was not clarified. In this study, the effects of ultrasonic vibration on the structure and properties of starch before and after the germination of highland barley were analysed.

This study examined highland barley of the Zangqing 2000 strain after being treated with 40 kHz ultrasonic vibrations for 40 min, followed by germination at 30 °C and 80% relative humidity (RH). A study of the effects of ultrasound and germination treatments on the macroscopic morphology of starch and its fine/molecular structure provides a theoretical foundation for developing new starch sources.

2. Materials and methods

2.1. Materials

The “Zangqing 2000” highland barley seeds were obtained from the Agricultural Research Institute of the Tibet Academy of Agricultural and Animal Husbandry Sciences. In autumn 2021, seeds were collected from a single field and stored at room temperature (20 °C). The chemical reagents used in the experiment were all analytical grade and did not require further purification.

2.2. Germination of highland barley using ultrasound

The first step involved selecting full grains of highland barley free of impurities and mold, sterilising them using 5% hydrogen peroxide for 30 min, and then soaking them in 3 volumes per weight of deionised water at 30 °C for 8 h. Afterwards, the highland barley seeds were divided into three groups, and germination was carried out using different methods. As part of the first group, seeds were germinated for a certain time, and at each sampling time (0, 24, 48, 72 h), the sample was terminated for germination, after which ultrasound was introduced. In this stage of the process, samples are no longer germinating. In the second group, the seeds were sequentially pretreated with ultrasound and then germination. Ultrasound was only carried out at 0 h. Seeds were germinated immediately following the ultrasound and removed after each germination time. Furthermore, seeds germinated alone served as an experimental control. Table 2 provides a description of the samples and procedures used.

Table 2.

List of Highland barley germination treatments.

Treatment	Processing details			Nomenclature
	Ultrasound	Germination time (h)	Ultrasound
Germination alone	–	0	–	G0
		24		G24
		48		G48
		72		G72
Sequential germination and ultrasound treatment	–	0	40 kHz ultrasound at 165.5 W for 40 min	G0-U
		24		G24-U
		48		G48-U
		72		G72-U
Sequential ultrasound pretreatment and germination	40 kHz ultrasound at 165.5 W for 40 min	0	–	U-G0
		24		U-G24
		48		U-G48
		72		U-G72

Note: In the experiment, the G0-U treatment is identical to the U-G0 treatment.

Barley mixed with water was homogenised using an impact mill (MJ-PB40E253C, Guangdong), sieved through a 100-gauge mesh sieve, and left for 2 h. The samples were centrifuged at 9000 rpm for 15 min, and then the obtained starch precipitate was rinsed three times with 95% anhydrous alcohol and dried. In order to obtain the starch flour, the dried samples were sieved with a diameter of 100 µm [23].

A 30 L ultrasonic cleaner (KC-100A, Dongguan, China) was used for the ultrasonic treatment. The seeds were treated using ultrasound (40 kHz, 240 W) at room temperature for 40 min and 2 cm in height from the bottom. Because of the energy loss in the ultrasound system, the actual level of ultrasound energy in the medium was lower than the set output power. Using the calorimetric method [24], the ultrasound power was determined to be 165.5 W.

2.3. Analyses of the macroscopic and morphological characteristics

2.3.1. LF-Nmr

As described earlier [25], low-field nuclear magnetic resonance was conducted with minor modifications. The sample temperature was maintained at 32 ℃. Bottle samples (height, 50 mm; inner diameter, 25 mm) were placed inside NMR tubes which were then positioned in the center of the RF coil in the LF-NMR instrument (LF-NMR, NMI20-015V-I, Suzhou, China). Each sample was analysed three times in parallel using CPMG sequences. Sequence parameters were as follows: magnet temperature, 32 °C; 90° pulse width, 15 μs; 180° pulse width, 29.52 μs; analog gain DRGI, 20; digital gain width, 3; main sampling frequency, 21 MHz; offset frequency, 264675.52 Hz; repeated sampling waiting time, 400; echo number NECH, 12000.

2.3.2. Sem

The morphology of starch was assessed using scanning electron microscopy (SEM). A conductive double-sided tape was applied to the sample table to distribute the dried starch uniformly. The sample was then examined under a SU8020 SEM (Hitachi, Tokyo, Japan) at a magnification of 1000× [26].

2.3.3. Particle size distribution

Sample suspensions (0.2 g in 0.8 ml of distilled water) were analysed using a laser particle size analyser (Malvern 3000 Malvern, UK) with shading set between 15% and 20% [27].

2.4. Molecular structure

2.4.1. Ftir

A spectrophotometer (Nicolet IS10, Waltham, MA, USA) was used to record FTIR spectra, with a frequency range between 4000 and 400 cm⁻¹ [28].

2.4.2. Xrd

X-ray diffraction was conducted using a D8 Advance XRD instrument (Bruker D8 ADVANCE, Karlsruhe, Germany). This diffractometer was equipped with a Cu-Kα radiation source and operated at 40 kV and 30 mA [29].

2.5. Fine structure analysis

2.5.1. Molecular weight distribution

SEC-MALLS-RI was used to examine the molecular weight distribution of the samples. A DAWN HELEOS-II laser photometer was used to measure the molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (Mw/Mn) of the samples (He-Ne laser, λ = 663.7 nm, Wyatt Technology Co., Santa Barbara, CA, USA). Three columns (300 × 8 mm, Shodex OH-pak SB-805, 804 and 803; Showa Denko K.K., Tokyo, Japan) were used simultaneously at 60 °C and 0.3 ml/min. In order to determine fraction concentrations and dn/dc values, a differential refractive index detector was used (Optilab T-rEX, Wyatt Technology Co., Santa Barbara, CA, USA). DMSO fractions showed a dn/dc value of 0.07 ml/g [30], [31].

2.5.2. Amylopectin distribution

Molecular weights were determined using SEC-MALLS-RI. Debranched starch molecular weights were determined using the differential refractive index detector described above using two tandem columns (300 × 8 mm, Shodex OH-pak SB-805 and 803) at 60 ℃ and 0.3 ml/min. Dextrans of known molecular weights were used to calibrate the columns (342; 3650; 21 000; 131 400; 610 500; 821 700; 3 755 000) [31], [32], [33].

2.5.3. Analysis of the distribution of amylopectin chain lengths

Amylopectin chain length distribution was analysed by high-performance anion-exchange chromatography (HPAEC) with a CarboPac PA-100 anion-exchange column (4.0 × 250 mm; Dionex) using a pulsed amperometric detector (PAD; Dionex ICS 5000 system) [34], [35]. In this experiment, the following parameters were employed: flow rate, 0.4 ml/min; injection volume, 5 μl; solvent system, 0.2 M NaOH (0.2 M NaOH, 0.2 M NaAc); gradient program, 90:10 V/V at 0 min, 90:10 V/V at 10 min, 40:60 V/V at 30 min, 40:60 V/V at 50 min, 90:10 V/V at 50.1 min, 90:10 V/V at 60 min.

2.5.4. Statistical analysis

A two-way ANOVA test and Tukey's test were used for statistical analysis. The data are presented as mean + standard deviation (SD), and statistical significance was assessed using SPSS 16.0 (SPSS Inc., USA). The difference is statistically significant if p < 0.05.

3. Results and analysis

3.1. Analysis of macroscopic and morphological characteristics

3.1.1. Evaluation of water migration and macroscopic images in highland barley seeds under a variety of treatment conditions

In order to study the migration of water, the hydrogen proton signal of internal water was monitored using LF-NMR. In the pseudo-color map, the red color (high brightness) represents regions with a high proton density and a higher water content. By contrast, the blue region (low brightness) indicates a low proton density and water content [36]. Fig. 1 illustrates the migration of water contents and macroscopic images under various treatments. A gradual increase in the internal water content of highland barley was observed during the germination treatment. Ultrasonic treatment at 0 h (i.e., G0-U and U-G0) significantly increased the moisture content of the seeds. In the entire germination process, the combined ultrasound-germination treatment resulted in significantly higher water content than the germination treatment alone.

Fig. 1.

Low-field nuclear magnetic resonance pseudo-color map and macroscopic images of treated samples Note: G0, G24, G48, and G72 are germinate alone for 0 h, 24 h, 48 h and 72 h without ultrasound; G0-U, G24-U, G48-U and G72-U are sequential germination for 0 h, 24 h, 48 h and 72 h, and ultrasound alone; U-G0, U-G24, U-G48 and U-G72 are ultrasound pretreatment, and then germination separately for 0 h, 24 h, 48 h and 72 h.

As can be seen in Fig. 1 (G0), highland barley was still dormant at 0 h after germination. A gradual increase in the water signal from G0 to G72 occurs as the germination time increases and the area gradually expands. These results are consistent with those obtained from the germination of mung beans [37]. Fig. 1 illustrates that the water content of U-G is greater than that of sequential germination and ultrasound treatment. Furthermore, when analysing Fig. 1 longitudinally, it is evident that the water content groups (red areas) are arranged in descending order as follows: U-G > G-U > G. This indicates that sequential ultrasound pretreatment and germination are superior to sequential germination and ultrasound treatment in any germination period (0–72 h). Ding et al. [20] demonstrated that ultrasound pretreatment could alter the surface microstructure of rice and facilitate better water transfer.

Like water migration trends, the shoot length of highland barley seeds increases with germination time. The shoot length of U-G is greater after 48 h of germination than that of sequential germination and ultrasound treatment. Andriamparany et al. [38] concluded that ultrasound treatment could break seed dormancy and promote germination.

3.1.2. Surface morphology of highland barley starch after different treatments

Different sources of starch can produce different shapes, but the most common is spherical or ellipsoidal [39]. At 0 h of germination (G0), the macroscopic morphology of the starch can be seen in Fig. 2; the surface and edges of the starch are smooth and rounded. Granules of starch G0-U/U-G0 were more sparse following ultrasound. In addition, it is observed that granules collapse after germination and become rougher over time. After the combined ultrasound and germination treatment, the surface roughness is more obvious, and the starch granules appear larger than after germination alone. This can be attributed to shear forces, microjets, and localised heating generated by sonication, which augmented sprouting effects on starch [40], [41].

Fig. 2.

SEM images of the treated samples. Note: The red arrow represents changes in starch under the corresponding treatment conditions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

A combination of ultrasound and germination treatment led to the breakdown of some of the larger starch granules into smaller granules that were either adsorbed by the larger granules or dispersed around the larger granules. Cavitation may occur in water solutions due to ultrasound. A bubble forms, which ruptures with increased shear forces within the granules, disintegrating the larger granules [42]. Cavitation has been observed in grooves on granular surfaces resulting from various grain germination [43]. In addition, these grooves may also be the result of partial hydrolytic degradation during germination, as reported earlier [44]. After sequential germination and ultrasound treatment of highland barley, the starch granules' surface was smoother than from germination alone.

Additionally, some small granules are stacked together to form larger starch agglomerates after G-U. Despite this, the degree of change in surface morphology was not greater than that produced by sequential ultrasound pretreatment and germination. There is a small difference in the roughness of the starch surface after sequential ultrasound pretreatment and germination. In contrast, the surrounding adsorption and stacking are very strong, suggesting that ultrasound reduces the hydrolysis of starch.

While starch morphology collapsed and became rough after germination, the combined ultrasound and germination exhibited a more significant effect and U-G > G-U.

3.1.3. Distribution of particle sizes in highland barley starch after different treatments

Fig. 3 exhibits that the particle size distribution significantly increased after germination and after all combination treatments. It is shown that the particle sizes of highland barley starch granules were larger after germination at 24 h, 48 h, and 72 h than at 0 h. These findings follow the observations of Gao et al. [45]; the particle sizes of pea starch also increased after germination treatment. In contrast, an increase in starch agglomeration would also increase particle size. It is also noteworthy that the G-U and U-G groups demonstrated the same trend with germination; starch particle size increased compared with germination under sequential ultrasound pretreatment and germination at 0 h. However, U-G treatment showed significantly larger size distributions than G-U treatment at 24 h, 48 h, and 72 h. These findings suggest that the G-U group is superior to the G group. This may be due to the mechanical, thermal and cavitation effects of ultrasonic waves, which strengthen the intermolecular force, leading to the aggregation of starch granules and the increase of particle size [46]. As discussed in Section 3.1.2, the cavitation effect caused by ultrasound contributes to the variation of the size distribution range.

Fig. 3.

Particle size distribution of highland barley starches subjected to different treatments Note: Particle size represents the size of granules. Volume represents the proportion of starch under this particle diameter. d (0.1), d (0.5), and d (0.9) indicate diameters that correspond to 10%, 50%, and 90% of the overall particle size distribution (0%–100%).

In the case of ultrasound combined with germination treatment, higher d (0.5) values were obtained compared to the treatment alone. There is evidence that ultrasound treatment causes cracks and destroys the integrity of seeds, thereby enhancing water binding, breaking seed dormancy, and facilitating germination. As a result of this process, starch absorbs water and swells, causing particles to enlarge [47]. The particle size of d (0.1) was the smallest, while that of d (0.9) was the largest in both germination alone and in combination with ultrasonication and germination. During 24 h, 48 h, and 72 h germination, the starch proportion of the G-U and U-G treatments was greater than that of germination alone under larger particle sizes, but the latter treatment was superior.

3.2. Molecular structure

3.2.1. Short-range ordering of highland barley starch after subjecting to different treatments

Short-range ordering of starch was examined with Fourier-transform infrared spectrometry. Fig. 4A illustrates the similar characteristic peaks in the infrared spectra of highland barley starch before and after G, G-U, and U-G with different treatment times, indicating that G, G-U, and U-G would not affect the functional groups of the starch samples. The absorption peaks at 3800–2800 cm⁻¹ can be attributed to the stretching vibrations of the hydroxyl (O-H) group and the polysaccharide's intermolecular or intramolecular hydrogen strength [48]. As the germination time extends, the absorption peak at 3000–2800 cm⁻¹ (the O-H stretching vibration peak) shows a red shifting. As a result of the enhanced hydrogen bonding in the system, the wave number shifts to a lower value [49]. U-G and G-U have stronger hydrogen bonds than G, and there is a greater shift in the O-H absorption peak in the U-G group than in the G-U group. During ultrasonic cavitation, hydrogen bonds are formed between the molecules of highland barley starch [46]. Compared to G-U and U-G, the peak value of U-G highland barley starch in the range of 3800–2800 cm⁻¹ changed more strongly than that of G-U. Using ultrasound enhanced the fluidity of highland barley starch and exposed more hydrophilic groups [50].

Fig. 4.

(A) FTIR spectra of the treated samples; (B) short-term changes in the crystal structure; (C) R_1047/1022 and R_1022/995 of the treated samples.

In starch, the fingerprint area is 1500–900 cm⁻¹, which can be used to assess subtle changes in its molecular structure. In this spectral region, the typical vibrational absorption peaks (Fig. 4B) appear at 1047 cm⁻¹, 1022 cm⁻¹, and 995 cm⁻¹. As a measure of the short-range structural order of the double helix in starch granules, it is possible to calculate the ratio between absorption peaks at R_1047/1022 and R_1022/995 [51]. An increase in the ratio of absorption peaks at R_1047/1022 indicates a higher short-range order. As can be seen in Fig. 4C, there is no significant difference among the different groups of R_1047/1022. It has been suggested that the decrease in the ratio of R_1047/1022 between G0 and G48h is related to the disintegration of the double helix structure in the crystalline region of germinated highland barley starch, which is in agreement with the findings of Liu et al. [52]. Further, the combined ultrasound-germination treatment increased the ratio of R_1047/1022, which suggests that G-U and U-G treatment can improve the crystallinity of starch with the extension of germinating time, and the effect of G-U treatment was more pronounced.

As a result of germination treatment, R_1022/995 was increased, indicating that the order of the double helix in starch granules decreased. As a result of the decrease in the R_1022/995 ratio of G-U and U-G, the degree of order of the double helix in starch granules has increased. Highland barley starch has a peak strength ratio of 0.757–0.943 at 1022 cm⁻¹/995 cm⁻¹, with G72-U being the lowest and G0-U being the highest, indicating the order degree of G72-U. At 1022 cm⁻¹/995 cm⁻¹, the ratio of peak strength for U-G highland barley starch is 0.766–0.943, with the lowest ratio being U-G48 and the highest ratio being U-G0, indicating the order degree of U-G48 is the lowest and the highest of U-G0. Additionally, it indicates that long-term G-U and U-G could weaken the short-term order of starch samples. According to Monroy et al. [42], the ordered structure of cassava starch was reduced following ultrasonic modification, resulting in loosely arranged starch granules following germination treatment. As a result of the ultrasonic treatment, the combination of starch chains was destroyed, and the short-range molecular order was reduced [53]. At the same time, the damage and degradation of rice starch molecules were induced by ultrasound, which affected the content of amylose and amylopectin [54]. Based on the above results, G-U highland barley starch possesses a higher surface order than U-G barley starch. It may be that the starch particles are eroded by ultrasonic treatment, resulting in broken starch chains and loose structures within the crystallisation zone.

3.2.2. Long-range ordering of highland barley starch subjected to different treatments

X-ray diffractometry was used to measure the spectral characteristics and relative crystallinity of starch samples after being subjected to different treatments. The diffraction characteristics of the samples were observed at 2θ of 15°, 17°, 18°, and 23°. A lower relative crystallinity is observed for G0-U/U-G0 than for G0. Germination alone or combined ultrasound-germination did not significantly alter the diffraction patterns of highland barley starch (Fig. 5B). According to Fig. 5A, the peak shapes did not change with treatment, and the crystallinity is of type A according to the results of Liang et al. [4] and You et al. [55]. In the combined ultrasound-germination treatment, the relative crystallinity of the starch was higher, while in the germination alone, it was lower. As a result of the degradation of α-amylase during starch germination, a change in the proportion of branched chains was observed, resulting in a decrease in relative crystallinity [3]. With sequential germination and ultrasound treatment, the relative crystallinity of starch increased with germination time. The relative crystallinity of starch was also increased following sequential ultrasound pretreatment and germination treatment. In the first instance, when barley germinates, starch hydrolase activity increases, accelerating the hydrolysis of starch and affecting the noncrystalline zone [20], [56]. Meanwhile, the stimulated buds grew longer and longer (Fig. 1), and the germinating group performed significantly worse than the ultrasound group. It has been reported that ultrasound treatment increased water uptake by switchgrass seeds during early absorption and may also have increased the level of endogenous plant hormones in plant tissues [20], [57]. Possibly, ultrasound promotes germination efficiency and further enhances crystallinity to some extent. According to Liang et al. [4], straight-chain starch may rearrange in both crystalline and noncrystalline regions after ultrasound treatment, resulting in a stronger ordered structure.

Fig. 5.

(A) X-ray diffraction patterns of the treated samples; (B) Relative crystallinity of the treated samples.

Thus, the ultrasound treatment increased the relative crystallinity of barley starch compared to germination alone and resulted in a tightercrystal structure. Ultrasound has a significant mechanical and cavitation effect on seeds before germination (or is more effective than germination first). Generally, starch crystallinity decreased after the barley was germinated alone. Starch crystallinity in the G-U treatment increased, but not significantly, as in the U-G treatment. As a result, ultrasound enhances starch's crystallinity to some extent.

3.3. Fine structure analysis

3.3.1. Distribution of molecular weight of highland barley starch following different treatments

In studying the molecular structure of starch, the molecular weight (Mw) is an important parameter since it directly influences its properties and applications [58]. The Mws of G0, G24, G48, and G72 in germination treatment were 2.48 × 10⁴ g/mol, 2.23 × 10⁴ g/mol, 2.38 × 10⁴ g/mol, and 2.27 × 10⁴ g/mol, respectively. Mws increased after combined ultrasound-germination treatment (2.47 × 10⁴ g/mol − 2.73 × 10⁴ g/mol); however, U-G values were always greater than G-U. It was demonstrated by You [55] and Pinkaew [59] that the enzymatic degradation of highly branched chains to less branched chains was responsible for the reduction in Mw after germination. The change in the number-average Mw of starch after sequential germination and ultrasound indicated starch degradation, corresponding to the breakdown of bonds within the starch molecule and reducing its Mw, which is in agreement with the results of Goni [60] and You [55].

The polydispersity coefficient d (Mw/Mn or Mz/Mw) can describe the polydispersity of starch molecular weights [61]. Larger d values (d > 1) indicate a greater molecular weight distribution or particle size difference. A study of the result of germination alone treatment on G24 showed the highest Mw distribution inhomogeneity (d = 2.35 × 10 ± 0.09), while G72 starch showed the lowest Mw distribution inhomogeneity (Table 3). After sequential ultrasound pretreatment and germination, an increase in the branched-chain Rz values was observed, which agrees with d. These results also indicated that the granules of starch were more inhomogeneous. As a result of sequential ultrasound pretreatment and germination, Rz also increased with germination, exhibiting the same trend as d, but the homogeneity of starch granules decreased. From Table 3, it is evident that the Mw of sequential germination and ultrasound increased from 2.47 × 10⁴ to 2.50 × 10⁴, and the Mw of sequential ultrasound pretreatment and germination increased from 2.47 × 10⁴ to 2.73 × 10⁴. Moreover, both groups of samples showed an increasing trend with germination time. Again, this indicates that ultrasound pretreatment impacts barley starch more than simultaneous germination and ultrasound. In general, the Mw of germinating alone shows a downward trend. At the same time, the Mw of sequential ultrasound pretreatment and germination at any time is higher than that of sequential germination and ultrasound.

Table 3.

Molecular weights of the treated samples.

Samples	Mn	Mp	Mw	Mz	d (Mw/Mn)	Rz
G0	1.08 × 104 ± 0.07d	4.72 × 104 ± 0.08d	2.48 × 104 ± 0.08d	4.11 × 104 ± 0.10d	2.29 ± 0.10b	77.7 ± 0.17b
G24	9.50 × 103 ± 0.06a	4.56 × 104 ± 0.08b	2.23 × 104 ± 0.07a	3.87 × 104 ± 0.16b	2.35 ± 0.09d	77.8 ± 0.16b
G48	1.03 × 104 ± 0.06c	4.63 × 104 ± 0.07c	2.38 × 104 ± 0.07c	4.00 × 104 ± 0.16c	2.30 ± 0.09c	78.4 ± 0.16b
G72	9.91 × 103 ± 0.06b	4.36 × 104 ± 0.07a	2.27 × 104 ± 0.07b	3.79 × 104 ± 0.15a	2.29 ± 0.09a	76.6 ± 0.16a
G0-U	1.10 × 104 ± 0.06d	4.62 × 104 ± 0.07a	2.47 × 104 ± 0.07c	4.07 × 104 ± 0.15b	2.24 ± 0.09a	77.7 ± 0.15a
G24-U	9.64 × 103 ± 0.06b	4.54 × 104 ± 0.07b	2.33 × 104 ± 0.06a	3.96 × 104 ± 0.15a	2.42 ± 0.08c	81.4 ± 0.13c
G48-U	9.37 × 103 ± 0.06a	4.77 × 104 ± 0.07d	2.36 × 104 ± 0.07b	4.10 × 104 ± 0.15c	2.51 ± 0.08d	85.1 ± 0.13d
G72-U	1.08 × 104 ± 0.07c	4.70 × 104 ± 0.08c	2.50 × 104 ± 0.08d	4.14 × 104 ± 0.19d	2.31 ± 0.10b	79.5 ± 0.17b
U-G0	1.10 × 104 ± 0.06b	4.62 × 104 ± 0.07a	2.47 × 104 ± 0.07a	4.07 × 104 ± 0.15a	2.24 ± 0.09a	77.7 ± 0.15a
U-G24	9.82 × 103 ± 0.06a	4.89 × 104 ± 0.07c	2.51 × 104 ± 0.08b	4.41 × 104 ± 0.17b	2.56 ± 0.10d	81.6 ± 0.16c
U-G48	1.23 × 104 ± 0.06d	4.94 × 104 ± 0.07d	2.78 × 104 ± 0.07d	4.46 × 104 ± 0.16d	2.26 ± 0.09b	79.0 ± 0.15b
U-G72	1.20 × 104 ± 0.06c	4.89 × 104 ± 0.07b	2.73 × 104 ± 0.07c	4.42 × 104 ± 0.16c	2.28 ± 0.08c	83.3 ± 0.14d

Note: Mn: number average molecular weight; Mw: heavy average molecular weight; Mz: z-average molecular weight; Mp: peak molecular weight; d (Mw/Mn): molecular weight distribution width index; Rz: mean square radius of gyration. Different letters indicate significant differences in the same treatment category (P < 0.05).

3.3.2. Distribution of chain length of straight-chain starch following different treatments

The Mw distribution of highland barley starch was evaluated using gel permeation chromatography (GPC). The three peaks in Fig. 6 (low, medium, and high Mw) are referred to as peaks 1, 2, and 3, respectively. Peaks 1 and 2 correspond to short (A and short B chains) and long (long B chains) branches of starch, respectively. Peak 3 represents straight-chain branches [62]. As the ratio of the areas of peaks 1 and 2 indicate the degree of branching in the starch, the higher the ratio, the greater the degree of branching [63].

Fig. 6.

Results of gel-permeation chromatography of the chain-length distribution in the treated samples.

Table 4 summarises the GPC parameters of highland barley starch for the 12 groups. Barley straight-chain starch content ranged from 15.95% to 19.74% across all treatments, with no significant differences between them. Short-chain starch ranged from 49.72% to 53.1%, while long-chain starch ranged from 19.39% to 21.79%. It is noted that G0, G0-U, and U-G0 possess the highest levels of short-chain amylopectin, while G72, G48-U, and U-G72 possess the lowest levels. Long-chain contents are highest in G48, G48-U, and U-G48, while they are lowest in G0, G24-U, and U-G0. The highest levels of amylose are found in the highland barley starches G72, G24-U, and U-G72, while the lowest levels are found in G0, U-G0, and G0-U.

Table 4.

GPC parameters of highland barley starch containing varying amounts of straight-chain amylose.

Samples	GPC peak area (%)
	Peak 1	Peak 2	Peak 3
G0	50.60 ± 2.02a	19.39 ± 0.78a	30.00 ± 1.20a
G24	50.13 ± 2.01a	19.64 ± 0.79a	30.23 ± 1.21a
G48	49.81 ± 1.99a	21.79 ± 0.87b	28.39 ± 1.14a
G72	49.72 ± 1.99a	19.58 ± 0.78a	30.70 ± 1.23a
G0-U	53.10 ± 2.12a	19.88 ± 0.80a	27.02 ± 1.08a
G24-U	50.26 ± 2.01a	19.45 ± 0.78a	30.28 ± 1.21b
G48-U	50.04 ± 2.00a	20.16 ± 0.81a	29.80 ± 1.19b
G72-U	50.61 ± 2.02a	20.00 ± 0.80a	29.40 ± 1.18b
U-G0	53.10 ± 2.12a	19.88 ± 0.80a	27.02 ± 1.08a
U-G24	51.60 ± 2.06a	20.90 ± 0.80a	27.49 ± 1.10a
U-G48	51.84 ± 2.07a	20.90 ± 0.84a	27.39 ± 1.10a
U-G72	50.89 ± 2.04a	20.25 ± 0.81a	28.86 ± 1.15a

Note: Different letters indicate significant differences within the same treatment category (P < 0.05).

The short-branched chains were found to be the most abundant in G0, G0-U, and U-G0, while the long-branched chains were found to be most abundant in G48, G48-U, and U-G48. The results of germination alone are consistent with those of sequential ultrasound pretreatment and germination, which shows that the treatment has no significant impact on the distribution of chain lengths of amylose. It has been shown that ultrasound significantly affects the chain length content of amylose after germination. Possibly, germination causes seed rupture, and ultrasonic treatment preferentially destroys amorphous areas, making it easier to attack amylose. Similar results have been reported by Luo et al. [64]. They concluded that ultrasonic treatment preferentially degrades amorphous areas and attacks amylose more readily than high-branched chain amylopectin.

3.3.3. Distribution of chain length of branched-chain starch in highland barley after different treatments

The chain-length distribution of branched starch was analysed using HPAEC. The chain-length distribution represents the degree of polymerisation and the relative contents of each chain type. The HPAEC [65] method can distinguish four side chain components based on the degree of polymerisation (DP), namely fa (DP 6–12), fbl (DP 13–24), fb2 (DP 25–36), and fb3 (DP > 36). The long chain (B2 + B3) is 18.68%-20.59% (Table 5). G0′s long chain (B2 + B3) ratio was 20.14, whereas G0-U/U-G0 after ultrasonic treatment was 19.39, indicating that the long chain was absorbed during ultrasonic treatment. In the germination process, the proportions of fa (DP 6–12) increased, while the proportions of fbl (DP 13–24), fb2 (DP 25–36) and fb3 (DP > 36) decreased. Furthermore, the A chain proportion of G0-U and U-G0 was higher than that of G0, while the long chain ratio (B2 + B3) was lower. A slight fluctuation in the average aggregation rate was observed following the combination of ultrasound and germination treatment. The average degree of polymerisation of G-U shows a wave-like fluctuation, whereas U-G decreases initially and then increases.

Table 5.

Distribution of crystal types and chain lengths in the treated samples.

Samples	G0	G24	G48	G72	G0-U	G24 -U	G48 -U	G72 -U	U-G0	U-G24	U-G48	U-G72
Crystal type	A	A	A	A	A	A	A	A	A	A	A	A
Crystallisation Yield(%)	26.02	26.25	25.04	19.36	19.68	26.26	25.94	24.70	19.68	24.39	24.52	27.64
A chain(%)	35.93	36.38	38.59	38.11	37.44	38.06	35.43	38.71	37.44	39.64	38.74	33.62
B1 chain(%)	43.92	43.94	42.19	42.58	43.2	42.45	44.44	41.97	43.20	41.68	42.22	45.76
B2 chain(%)	12.49	12.29	11.94	12.03	12.08	12.03	12.59	11.94	12.08	11.59	11.88	12.86
B3 chain(%)	7.65	7.38	7.30	7.27	7.31	7.46	7.54	7.40	7.31	7.09	7.13	7.73
B2 + B3	20.14	19.67	19.24	19.30	19.39	19.49	20.13	19.34	19.39	18.68	19.01	20.59
Average degree of polymerisation	18.37	18.20	17.89	17.96	18.06	18.03	18.38	17.93	18.06	17.70	17.82	18.66

Different letters within the same treatment category indicate significant differences (P < 0.05).

Fig. 7 shows that germination treatment reduced the number of long chains (B2 + B3) and their relative crystallinity. Veluppillai et al. [66] observed that amylase activity significantly increased in germinated brown rice between 0 and 9 days, possibly as a consequence of enzymatic degradation of the branched-chain starch. The molecular weight distribution is also consistent with this result. As a result of ultrasonication, starch's Mw (heavy average molecular weight) is also increased. It has been proposed that the cavitation effect of ultrasound may induce the release of α-amylase as it is likely to impact the enzyme's activity [21], [67], [68]. α-amylase may be capable of binding long-chain starch molecules in the early stages and may be preferred to interact with short-chain starch molecules in the later stages. Consequently, there were slight fluctuations in the average amount of polymerisation in sequential ultrasound pretreatment and germination, especially in the late stage (after 24 h), when the long chain (B2 + B3) was increasing.

Fig. 7.

Chain-length distribution of branched starch in the treated samples.

4. Conclusions

The study aimed to evaluate the effects of combining ultrasound and germination treatments on the structure of highland barley starch. In the combined ultrasound-germination treatment, Mws was improved, but the U-G value was higher than the G-U value. Furthermore, after sequential ultrasound pretreatment and germination, the degree of polymerisation of the branched starch chain lengths was altered slightly. In addition, FTIR and XRD measurements also revealed that sequential ultrasound pretreatment and germination increased hydrogen bonding in starch and its crystallinity. Based on the macroscopic morphology, it was found that the water content, surface roughness, and grain size distribution of the barley in the U-G group increased significantly.

The results of the ultrasound pretreatment are more obvious in terms of the macroscopic morphology, the molecular structure and the fine structure of highland barley starch. In the case of sequential germination and ultrasound treatment, ultrasound pretreatment offers more advantages. The second factor contributing to developing and utilising highland barley is its healthcare-related applications. Germination methods, combined ultrasound-germination methods, or other modification methods show potential for developing and utilising highland barley in the future. An investigation into the relationship between the GABA content and the structure of highland barley starch will be conducted in the future.

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.