Effects of ultra‐high pressure on the morphological and physicochemical properties of lily starch

Abstract In this study, starch extracted from lily bulbs were modified using an ultra‐high pressure (UHP) treatment at six different pressure levels (100, 200, 300, 400, 500, and 600 MPa). The effects of UHP treatment on the physicochemical and morphological properties of lily starch were investigated. The morphological observation revealed that UHP treatment led to particle expansion and aggregation. Compared with the native and lily starch treated at 100–500 MPa, the lily starch treated at 600 MPa exhibited almost completely disrupted morphology and a larger particle size, indicating nearly complete gelatinization of the starch. The relative crystallinity of the UHP‐treated starch remarkably reduced. Gelatinization temperatures via differential scanning calorimetry decreased with increasing pressure. The rapid viscoanalyzer results revealed that the lily starch treated with UHP at 600 MPa showed low values of peak viscosity, trough viscosity, breakdown, final viscosity, and setback. These results indicated that UHP was an effective physical modification method for lily starch, UHP treatment (600 MPa, 30 min) caused nearly complete gelatinization of lily starch, and lily starch modified using UHP might expand the application of lily in the food field.

change the structure of starch granules and endow its characteristic properties (Li et al., 2014;Piecyk et al., 2018). In the previous study, our laboratory modified lily starch by using heat-moisture and acid treatments and found that modified lily starches presented enhanced physicochemical properties and could be used in industrial-resistant starch and food ingredients . Starch depolymerization easily occurred in the acid treatment and was unconducive to the environment. Therefore, an environment-friendly modification method for lily starch should be developed.
Ultra-high pressure (UHP) treatment is a typical physical nonthermal modification method. It is regarded as a green and environment-friendly physical modification method and can be used for starch modification. In previous reports, the effects of UHP on different kinds of starch have been studied. Li et al. (2015) found that UHP treatment could promote water molecules to enter red adzuki bean starch granules, disrupt the crystalline structure, and reduce the thermal stability;  found that UHP-treated lotus seed starch showed lower retrogradation tendency compared to native starch; Larrea-Wachtendorff et al. (2019) obtained high viscosity and highly structured potato starch hydrogels through UHP-treated. Alvarez et al. (2014) found that the chickpea flour slurry product would exist better flow characteristics after modified by UHP-treated. Liu et al. (2018) found that the in vitro digestibility of pea starch treated with UHP was remarkable lower than that of the native starch. These studies have indicated that UHP treatment could change the structure and physicochemical properties of starch and endow starch with new functional properties. Different crystallite types of starch show diverse results after UHP treatment. A-type starch is the most sensitive to pressure, followed by C-and B-type starch (Kim et al., 2018). Sorghum (A-type) is completely gelatinized at 480-600 MPa .
The starch of mung bean (C-type) is completely gelatinized at 600 MPa (Li et al., 2011). Potato starch (B-type) is gelatinized at 800 MPa (Błaszczak et al., 2005). The gelation degree of starch increased with the increases in water content and temperature. Although the pressure is adequately high, it could also cause complete gelation of starch at room temperature .
However, to the best of our knowledge, no report is available on the physicochemical and morphological properties of lily starch treated with UHP. In this study, lily starch was treated at six UHP levels. The surface morphology, particle size, X-ray patterns, thermal properties, Fourier-transform infrared (FTIR) spectrum, and pasting properties of native lily and UHP-treated starch were investigated. The experimental results can provide references for the application of lily starch.

| Materials
The fresh bulbs (Lilium brownii var. viridulum Baker) used in this work were cultivated in Longhui County, Hunan Province, China. After harvesting, the bulbs were transported to the laboratory immediately. Bulbs with physical damage and pests were selected out.
Undamaged lily bulbs were peeled and washed with tap water, and then, the washed lily scales were placed into a wall breaker to homogenate for 2 min (v lily scales : v dstilled water = 1:2).

| Lily starch isolation
The lily bulb starch was extracted according to Zhang, Saleh, et al. (2020) with some modifications. The well-homogenized lily slurry was filtered with a 100 mesh nylon cloth, and the residue was rinsed repeatedly with distilled water until no more starch filtrate was released. The collected filtrate was centrifuged at 25°C at 5,000 × g for 10 min. The precipitated starch granules were then washed with 0.05 mol/L of NaOH and stirred every 30 min. The NaOH solution was replaced every 3 hr until the supernatant of the cleaning solution became colorless and the cleaning was stopped. The starch samples were freeze-dried, sifted through 100 mesh, and stored in a desiccator for further use.

| UHP treatment
Lily starch was subjected to UHP treatment (high-pressure press-type SHPP-8.8 L, Shanxi Sanshuihe Technology Co., Ltd.). The extracted starch was prepared into 15% (w/w) starch-water suspension and divided into seven equal parts. The suspension was packed into polythene bags, shaken thoroughly to move the bubbles of the vacuum bag, and sealed with a vacuum packer. The sealed samples were transferred into a pressure chamber and subjected to different pressure levels (100, 200, 300, 400, 500, and 600 MPa) at room temperature for 30 min. After UHP treatment, the samples were vacuum-filtered, and the supernatant was removed after centrifugation with 5,000 × g at 25°C for 10 min. The samples were freeze-dried, and each dried starch sample was pulverized with pestle and mortar and then kept in a desiccator at room temperature for further analysis.

| Polarized light microscopy (PLM)
Polarized and normal light microscopic images were recorded on a polarized light microscope (LEICA DM4500P; Leica Microsystems) by using the method of Guo, Zeng, Zhang, et al. (2015). A starch sample of 1 mg was dispersed in glass slides with glycerol and water (1:1, v/v). The PLM instrument was used to observe at 200× magnification under normal and polarized light conditions.

| Particle size determination
The particle size parameters of starch were measured using a laser diffraction particle size analyzer (LS-POP laser particle size analyzer, Omec Technology Co., Ltd.), as described by Li et al. (2019). In detail, 1.5 g of lily starch was evenly dispersed with 20 ml of distilled water, and then, the suspension was poured into the rotating container of the diffraction particle size analyzer with a shading ratio ranging from 8% to 15%. Refractive indices (dn/dc) of starch and water were set to 1.60 and 1.33, respectively. Volume particle size (D (4,3) ), surface particle size (D (3,2) ), D 10 , D 50, and D 90 were recorded. D (4,3) presents the particle diameter of volume, D (3, 2) presents the particle diameter of surface, and D 10 , D 50 , and D 90 represent the corresponding particle sizes which are smaller than 10%, 50%, and 90% of the sample particles, respectively.

| X-ray diffraction analysis
X-ray diffractograms of lily starch samples were obtained using an X-ray diffractometer (XRD-6000, Shimadzu) in accordance with the method of Ahmed et al. (2018) with some modifications. The measurement was operated at Cu-Kα (λ = 1.5418 nm), X-ray tube 40 kV, and voltage of 40 mA, the diffraction scanning angle was from 5° to 45° (2θ), the step size was 0.015°, and the scanning speed was 8°/ min. The relative crystallinity of lily starch was calculated using MDI Jade software.

| FTIR spectroscopy
The FTIR spectra of starch samples were recorded on an FTIR spectrophotometer (Model IRAffinity−1, Shimadzu) at room temperature, as described by Rafiq et al. (2016). Starch was mixed with dried KBr powder in a ratio of 1:100 (m/m) and pressed into transparent tablets under infrared light before measurement. KBr was scanned as background, and the spectra were recorded within the range of 400-4,000 cm −1 .

| Determination of thermal properties
The thermal properties of the lily starch samples were measured using a differential scanning calorimeter (Q2000-DSC, TA Instruments) in accordance with the method of Li et al. (2020). Each starch sample of 5.0 mg was accurately weighed into a differential scanning calorimetry (DSC) pan, and 10 μL of distilled water was added. Before the experiment, the samples were balanced at room temperature for 24 hr, with an empty aluminum pan as a reference.
The scanning temperature was from 30°C to 110°C, and the heating rate was 10°C/min. The thermal parameters, including onset temperature (T o ), peak temperature (T p ) and conclusion temperature (T c ), the gelatinization temperature range (ΔT r ), and the gelatinization enthalpy (ΔH), were recorded.

| Pasting properties
The test was performed as described by Zhang, Ma, et al. (2020) with some modifications. The pasting properties were analyzed using a rapid viscoanalyzer (RVA Super-4, Newport Scientific). Each sample (3.0 g, dry basis) was weighed into an RVA canister and added with 25 ml of distilled water. The slurry was then homogenized using a plastic paddle to avoid lump formation before the RVA run. The starch slurry was heated from 50°C to 95°C at 12°C/min and kept at 95°C for 2.5 min, then it was cooled to 50°C at the same rate with a paddle speed of 160 rpm. Pasting properties, including peak viscosity (PV), trough viscosity (TV), breakdown (BD), final viscosity (FV), setback (SB), peak time (PT), and pasting temperature (PT), were determined.

| Statistical analysis
All the tests were performed in triplicate. Data were expressed as mean ± standard deviations. Statistical analysis was conducted using SPSS20.0 for Windows, and data were analyzed using ANOVA with Duncan's multiple range tests (p < .05). ORIGIN 7.5 was also used for statistical analysis.

| Morphological properties
The microscopic images of the native and UHP-treated lily starches with SEM are shown in Figure 1 (500×). Native lily starches showed round-shaped, oval-shaped, and other irregular granules with a smooth surface; this result is similar to a previous result observed by Li et al. (2020). After UHP treatment with the pressure ranging from 100 MPa to 400 MPa, the appearance of starch granules did not change significantly, which implied that the starch structure cannot be disrupted under these pressures (Figure 1b-e). When the treatment pressure was further increased to 500 MPa, the surface of starch granules began to shrink, and starch granules presented some fragments and a tendency to expand (Figure 1f). These observations demonstrated that the structure of the lily starch granules started to lose when the treatment pressure was increased to 500 MPa. The structure of starch granules was almost entirely disrupted, and a gel-

| Light microscopy
The birefringence phenomenon of the native and UHP-treated starch SEM results also showed that the crystal structure of the particles was seriously damaged. This condition indicated that 600 MPa was the critical pressure for the complete gelation of lily starch.

| 3 Particle size distribution (PSD)
The PSD of the native and UHP-treated starch is shown in Figure 3.   (Liu, Guo, et al., 2016). The results showed that the effect of UHP on the morphology of starch granules was related to the pressure, with 600 MPa resulting in a transition of the lily native starch structure to gelatinized starch paste.

| X-ray diffraction
In accordance with the X-ray diffraction pattern, the starch can be divided into three categories: A-type (mainly exists in corn starch, with a strong diffraction peak at 2θ values of 15°, 17°, 18°, and 23°),

B-type (mainly exists in the tubers of plants rich in carbohydrates,
with a very strong diffraction peak at 17° and weak diffraction peaks at 20°, 22°, and 24°), and C-type (mainly exists in leguminous plants; it is a mixture of A-and B-type starch) (Liu, Guo, et al., 2016;Zobel, 1988). The native starch presented diffraction peaks at 14.92°, 17.02°, 19.46°, 22.24°, and 23.58° (Figure 4). The diffraction peak at 17.02° was very strong; hence, lily starch is a typical B-type starch. After the UHP treatment from 100 to 600 MPa, no changes in X-ray diffraction patterns could be observed, the disappearance of characteristic diffraction peak was not found, the change in crystal properties was mainly manifested as the change in diffraction peak strength, and the crystal type was still B-type.
This condition also suggested that the crystalline structure of starch was vulnerable to disruption at high pressure levels. In the previous research, potato starch also maintained a B-type diffraction pattern after UHP treatment (Błaszczak et al., 2005;McPherson & Jane, 1999) Sorghum (A-type) and mung bean starch (C-type) were completely gelatinized at 600 MPa, and X-rays showed that the starch granules gradually exhibited the diffraction pattern of B-type crystal Li et al., 2011). The B-type starch was insensitive to UHP compared with A-and C-type starch because it had a more open structure containing a hydrated helix core (Liu et al., 2010). Figure 5 shows the DSC diagram of the native and different UHPtreated lily starch granules. With increasing pressure, the endothermic peak was gradually shifted to an increased temperature and became weak. A similar phenomenon has been observed for lotus seed starch . Note: Means followed by the same small letter within a column are not significantly different (p < .05); D (4, 3) presents particle diameter of volume; D (3, 2) presents particle diameter of surface; D 10 , D 50 , and D 90 represent the corresponding particle size which is smaller than 10%, 50%, and 90% of the sample particles, respectively.

| Thermal properties
granules under different treatment conditions Liu, Yu, et al., 2009;Zeng et al., 2018). Relatively low pressure could not disrupt the double helices to coil state, the helices could only be separated side by side, and the helices could be dissociated into a single helix only when a certain pressure was reached. The pressure treatment of starch below 500 MPa disturbed the crystal structure of the lily starch, and 600 MPa was the critical pressure to disrupt the crystal structure. Pressure is an important factor in starch gelatinization.

| FTIR spectroscopy
Fourier-transform infrared spectroscopy of native and UHPtreated starches is presented in Figure 6.   , 2018). The intensity of peak at 2,100 cm −1 increased with UHP treatment at 0-500 MPa, thereby indicating that the amount of free water in the above starches increased. The peak was relatively gentle at 600 MPa, indicating that the content of free water in starch decreased significantly. The peak at 2,368 cm −1 was due to C-H stretching associated with ring methane hydrogen atoms (Rafiq et al., 2016). Substantial hydroxyl groups exist in natural starch, which induce intermolecular and intramolecular hydrogen bonds in the main chain of starch. The double-helix structure was held together by hydrogen bonds. However, in the gelation process, water penetration disrupted this structure, resulting in infrared spectral fluctuations in the relevant region. The bond at 2,927 cm −1 was related to the C-H stretching and bending vibration of methyl and methylene groups of polysaccharides (Wang, Xie, et al., 2020;Weerapoprasit & Prachayawarakorn, 2019

| Pasting properties
Variations in pasting properties of the native and UHP-treated samples are shown in Abbreviations: ND, not detected; T c , conclusion temperature; T o , onset temperature; T p , peak temperature; ΔH, enthalpy of gelatinization; ΔT r , gelatinization temperature range (ΔT r = T c -T o ).

F I G U R E 6
Fourier-transform infrared spectra of the native and ultra-high pressure treated lily starch

| CON CLUS IONS
In this article, lily starch was physically modified using UHP. UHP