Effects of plasma-activated water combined with ultrasonic treatment of corn starch on structural, thermal, physicochemical, functional, and pasting properties

Highlights

• •A combination of PAW and UL (PUL) was applied for corn starch modification.
• •Surface destruction and increased in short-range order and crystallinity were observed.
• •PUL increased the gelatinization parameters and reduced retrogradation of starch.
• •PUL enhanced water absorption, swelling power, and color properties of starch.
• •The starch subjected to dual modification exhibited the most ideal properties.

Abstract

In this study, corn starch was used as the raw material, and modified starch was prepared using a method combining plasma-activated water and ultrasound treatment (PUL). This method was compared with treatments using plasma-activated water (PAW) and ultrasound (UL) alone. The structure, thermal, physicochemical, pasting, and functional properties of the native and treated starches were evaluated. The results indicated that PAW and UL treatments did not alter the shape of the starch granules but caused some surface damage. The PUL treatment increased the starch gelatinization temperature and enthalpy (from 11.22 J/g to 13.13 J/g), as well as its relative crystallinity (increased by 0.51 %), gel hardness (increased by 16.19 %) compared to untreated starch, without inducing a crystalline transition. The PUL treatment resulted in a whitening of the samples. The dual treatment enhanced the thermal stability of the starch paste, which can be attributed to the synergistic effect between PAW and ultrasound (PAW can modify the starch structure at a molecular level, while ultrasound can further disrupt the granule weak crystalline structures, leading to improved thermal properties). Furthermore, FTIR results suggested significant changes in the functional groups related to the water-binding capacity of starch, and the order of the double-helical structure was disrupted. The findings of this study suggest that PUL treatment is a promising new green modification technique for improving the starch structure and enhancing starch properties. However, further research is needed to tailor the approach based on the specific properties of the raw material.

1. Introduction

Starch is not only a primary nutrient in human food and animal feed but also an essential raw material in various fields such as food, pharmaceuticals, textiles, papermaking, and the chemical industry [1], it is widely used in both food and non-food industries [2]. As a native polysaccharide, it has garnered widespread attention for its excellent thickening, adhesive, gelatinization, gelling, and film-forming capabilities [3], however, native starch is characterized by high viscosity, poor solubility, low heat/acid resistance, low water absorption, and a tendency to retrograde, which limits its extensive application in processing [4]. Therefore, it is necessary to modify starch to overcome these shortcomings and increase its applications in various industries.

The modification of starch typically involves physical, chemical, and biotechnological methods [5], [6]. The high cost, potential food safety and environmental issues associated with chemical modification, and the relatively limited efficacy and application scope as well as the high cost of biotechnological methods, have limited their application. Physical modification has attracted widespread attention for its simplicity and economy, and the absence of chemical and biological reagent issues [7]. Among various physical modification techniques, cold plasma (CP) and ultrasonic technology are favored for their safety, speed, and eco-friendliness [8], [9], [10], [11]. CP is referred to as the fourth state of matter and is a significant process for altering the physicochemical properties of materials [12]. It operates under mild, non-toxic, environmentally friendly conditions with high processing efficiency. CP modifies the surface of starch through methods such as bombardment with high-energy active particles, radiation, and free radical oxidation. This can enhance the hydrophilicity and adhesive quality of starch without affecting its overall properties. This method expands the application range of starch and represents a promising technology [9]. Ge et al. [13] found that CP treatment resulted in a decrease in molecular weight, long chain ratio, crystallinity, and enthalpy of red bean starch, which led to more processing properties of the starch without disrupting the granule morphology. Although various authors have studied gaseous plasma as a strategy for modifying starch [11], [14], [15], there are certain limitations. Direct treatment of starch with gaseous plasma leads to a rapid reaction that is difficult to control and can inflict some damage on the starch. Additionally, direct contact between gaseous plasma and starch can cause uneven treatment and scorching [15]. Plasma-activated water (PAW) is generated by exposing water to a plasma discharge environment that contains reactive nitrogen species (RNS) and reactive oxygen species (ROS) such as hydrogen peroxide, ozone, nitrite, and nitrate [16], these reactive species can interact with starch molecules, altering their structure and properties. Such changes include the breaking or reorganizing of starch molecular chains, enhancements in crystalline and double-helical structures, and improvements in thermal stability, gel strength, and viscosity [17]. Applying PAW technology offers a new perspective for functionalizing starch and its derivatives, opening up new fields in starch science and technology research.

Ultrasonic technology, as a simple, fast, and efficient physical modification technique, has been proven to alter the physicochemical properties of starch effectively [18]. Ultrasonic treatment primarily affects the amorphous regions of starch granules, generating a microporous structure within the granules without compromising the integrity, size, internal morphology, and crystallinity [[19], [20]]. The ultrasonic process uses energy generated by sound waves with frequencies exceeding 20 kHz to agitate the suspension medium [21]. The process can disrupt the hydrogen bonds of starch at the molecular level, rearranging its crystalline structure, thereby enhancing the starch's solubility, pasting properties, and film-forming ability. Rahaman et al. [22] subjected starch to ultrasonic treatment at a frequency of 40 kHz for 20 min. After the ultrasonic treatment, the crystalline structure of the starch was disrupted, the crystallinity decreased, and grooves and notches appeared on the surface of the starch granules. Ultrasound can alter the physicochemical and functional properties of starch, offering advantages such as high selectivity towards specific molecular structure and chemical bonds, good functional properties, minimal chemical usage, and short processing times [23].

In some studies within both food and non-food industries, it has been shown that the sole modification of starch can yield less optimal results compared to combined modification techniques [24]. Hence, dual modification has been introduced to enhance the various properties of starch. Due to the rearrangement of starch granules, dual modification can significantly change the physicochemical properties of starch [25]. Combining ultrasonic and PAW technologies to modify corn starch aims to develop starch materials with improved performance, meeting the stricter requirements of specific application areas. Recently, ultrasound and cold plasma techniques have been applied to treat starch from different sources (such as potato starch, corn starch, and mung bean starch) to investigate their effects on their structure, physicochemical properties, functional properties, and thermal properties [[18], [25], [26]]. To our knowledge, there is still a lack of insight into the effects and mechanisms of PAW combined with ultrasound treatment on the structural, physicochemical, and functional properties of corn starch. Therefore, the effects of PAW and ultrasound treatment on the structural, thermal, physicochemical, functional, and pasting properties and functional groups of corn starch were investigated. This study provides deeper insights into the effects of starch modification through the combined use of PAW and ultrasound treatment, as well as the potential for scaling up this technology in the industrial production of starch-containing materials. This multi-technological fusion approach promises to bring innovative solutions to starch science research and industrial applications, facilitating the transformation of starch materials towards functionalization and enhanced performance.

2. Materials and methods

2.1. Materials

Corn starch (purity ≥ 98 %) was purchased from Yuan Ye Biotechnology Co., Ltd. (Shanghai, China). Other analytical grade chemical reagents were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. (Beijing, China).

2.2. Preparation of PAW

In this study, the PAW was generated using a modified dielectric barrier discharge (DBD) plasma system (Nanjing Suman Electronics Co., Ltd., China) [27], which consists of a plasma generator (with a fixed discharge frequency of 10 kHz), a pulse generator, a DBD chamber, a voltage booster, and a digital oscilloscope. As shown in Fig. 1, the distance between the plasma source and water was set to 5 mm, with the activation time specifically established at 10 min for each treatment of 50 mL of distilled water. Immediately following plasma activation, measurements of the hydrogen peroxide concentration, nitrite concentration, nitrate concentration, pH, oxidation–reduction potential (ORP), and electrical conductivity (EC) in the plasma-activated water (PAW) were conducted. These parameters were also assessed in the water before plasma activation. The contents of H₂O₂ in PAW samples were measured with the ferrous oxidation-xylenol orange (FOX) assay [28]. The concentrations of nitrite and nitrate were measured using spectrophotometry [29]. The pH and ORP of the PAW were measured using a pH/ORP meter (FE20, Mettler-Toledo Instruments Co., Ltd., Shanghai, China). The EC was determined with a conductivity meter (Leici DDS-307A, Shanghai, China). All measurements were conducted immediately after treatment and repeated at least three times.

Fig. 1.

A schematic diagram of the DBD plasma system and the experimental arrangement.

Voltage (V) and current (i) during the plasma discharge process were measured using a high-voltage probe and a current probe, respectively, both connected to a digital oscilloscope (TBS 1000C, Tektronix, USA). The average discharge power (P) was calculated using the following equation:

$$P\text{= 1/}T{\int }_0^{T}i(t)V(t)dt$$ (1)

Where T represents the applied voltage period.

2.3. Preparation of samples

2.3.1. PAW treatment

Corn starch was directly mixed with PAW in a 1:2 (w/v) ratio for 20 min with continuous mixing. Using a magnetic stirrer (Heidolph MR Hei Standard, Schwabach, Germany), the mixture of corn starch and PAW was stirred at 500 rpm. This treatment condition was determined based on preliminary testing to determine the maximum efficiency of the PAW (data not reported). The samples without any treatment were considered native corn starch and named the control group (CK). Starch mixed with distilled water (SDW) is also considered a native starch but involves a mixing step with distilled water to distinguish any effects of water mixing from the untreated starch. The PAW-treated starch is named PAW.

2.3.2. Ultrasound treatment

A 300 mL starch suspension was subjected to ultrasonic irradiation using an ultrasonic probe processor (Scientz-IID, Ningbo Scientz Biotechnology Co., Ningbo, China) at 25 °C. The ultrasonicator, equipped with an ultrasonic horn, was placed in a 500 mL glass beaker and operated in pulse mode (3 s on and 4 s off) to mitigate the thermal and overheating effects on the starch, with a continuous duration of 40 min, a rated power of 300 W, and an ultrasonic frequency of 25 kHz. The probe, featuring a 10 mm diameter vibrating titanium tip, was immersed below 30 mm from the surface of the starch suspension. During the ultrasonic treatment, the beaker was submerged in a constant temperature water bath (25 ± 0.5 °C). The samples treated in this manner were designated as UL.

2.3.3. Dual modification

The 300 mL sample treated with PAW (Section 2.3.1) was subjected to ultrasound under the conditions described in Section 2.3.2, and the samples were labeled as PUL. All treated samples were dried overnight in an oven (DHG-9240A, Shanghai Jinghong Experimental Equipment Co., Ltd., Shanghai, China) at 45 °C, ground, sieved, and stored for further analysis.

2.4. Color characteristics

The color of corn starch was determined using a hand-held colorimeter (RM200QC, X-Rite, Germany). L* denotes brightness (0-black, 100-white), a* denotes green (−)/red (+), and b* denotes blue (−)/yellow (+). The total color difference (ΔE), whiteness index (WI), and chroma (C) are calculated by the following equations [30].

$$\mathrm{\Delta }E\sqrt{{(L^{\ast }-L_0^{\ast })}^2+{(a^{\ast }-a_0^{\ast })}^2+{(b^{\ast }-b_0^{\ast })}^2}$$ (2)

$$\mathit{WI}=\sqrt{{(L^{\ast }-L_0^{\ast })}^2}$$ (3)

$$C=\sqrt{a^{\ast 2}+b^{\ast 2}}$$ (4)

L₀^∗, a₀^∗, and b₀^∗ were determined from CK samples.

2.5. Pasting properties

The pasting properties of corn starch were measured using a rapid viscosity analyzer (RVA TecMaster, Perten Instruments, Inc., Hägersten, Sweden). Briefly, 3.68 g of corn starch in 25 mL of deionized water was dispersed into the RVA canister. Before placing the canister in the apparatus, the mixture is first stirred with a plastic paddle. The samples were held at 50 °C for 1 min, heated to 95 °C at 7.5 °C/min, held at 95 °C for 3 min, then cooled to 50 °C at 11.25 °C/min and held at 50 °C for 2 min [31]. The rotational speed was kept at 960 rpm for the first 10 s, and the rest was performed at 160 rpm. Paste temperature (PT), peak viscosity (PV), breakdown viscosity (BV), setback viscosity (SV), final viscosity (FV), and trough viscosity (TV) were recorded.

2.6. Morphological characterization

2.6.1. Scanning electron microscopy (SEM)

The microstructure of corn starch was observed using the SU3500 scanning electron microscope from Hitachi Co., Tokyo, Japan. The sample was observed under vacuum mode using an accelerating voltage of 15 kV. Before observation, the sample was dispersed on a sample holder and sputter-coated with gold.

2.6.2. Confocal laser scanning microscopy (CLSM) imaging

Starch samples (10 mg) were mixed with 10 mmol/L 8-aminophenyl-1,3,6-trisulfonate trisodium salt in acetic acid solution (15 μL) and 1 mol/L sodium cyanoborohydride (15 μL), and then allowed to stabilize at room temperature for 24 h. The stained starch samples were washed five times with deionized water (1 mL) and suspended in a 100 μL mixture of 50 % glycerol and water. A drop of this suspension was placed in a confocal laser scanning microscope (ZEISS LSM900, Germany) to observe the microstructure of the starch granules [32].

2.6.3. Optical microscope (OM) observation

Using a B × 53 light microscope (Motic Olympus, China), the micromorphology and Maltese cross of starch were observed, and images were captured using a SPOT Insight camera with 20 × magnification.

2.7. Texture profile analyses (TPA)

Corn starch at a 10 % (m/v) suspension was gelatinized in a boiling water bath for 20 min, after which the gelatinized corn starch was transferred into a cylindrical mold measuring 20 × 20 mm and cooled at 4 °C for 24 h. The cooled corn starch was then removed from the mold and allowed to stand at room temperature (25 °C) for 1.5 h before the strength of the starch gel was measured using a texture analyzer (TA-XT plus, Stable Micro System Ltd., UK). Subsequently, the hardness and elasticity were used by Kuo et al. [33], with some modifications. In simple terms, the samples were compressed twice using a cylindrical aluminum probe with a diameter of 36 mm to 50 % of their original height. The speed was set to 1 mm/s before, during, and after the test, with a trigger force of 0.01 N. The interval between the first and second compression was maintained at 5 s. An appropriately sized plastic film was placed between the gel samples and the probe to prevent the gel samples from adhering to the probe.

2.8. Starch gelatinization

The gelatinization properties of corn starch were measured using a differential scanning calorimeter (DSC214, Netzsch, Germany). Starch (3 mg) was placed in an aluminum pan and mixed with deionized water (9 μL). The pan was sealed and equilibrated overnight at room temperature. The sample tray was equilibrated at 25 °C for 2 min before the test, then heated from 25 °C to 120 °C at a rate of 10 °C/min [34]. The onset temperature (T_o), peak temperature (T_p), conclusion temperature (T_c), and the gelatinization enthalpy (ΔH) were recorded.

2.9. X-ray diffraction (XRD)

Based on the method by Chen et al. [35], the crystalline structure of corn starch was determined using an X-ray diffractometer (XD-3, Beijing Purkinje General Instrument Co., Ltd., China). During the experiment, nickel-filtered CuKα radiation was applied at a voltage of 36 kV and a current of 20 mA. The scanning range was from 5° to 40°, with a rate of 2°/min and a sampling interval of 0.02°. The relative crystallinity (%) was calculated using MDI Jade 6 software (Materials Data Inc., Livermore, USA).

2.10. Fourier transform infrared (FTIR) spectroscopy

The characterization of functional groups and chemical bonds in corn starch was determined using a PerkinElmer spectrum 100 FTIR spectrometer (PerkinElmer Co.,

Ltd., Massachusetts, USA). Corn starch (2 mg) and KBr (200 mg) were thoroughly ground in an agate mortar and then pressed into a transparent sheet using a mold provided by the FTIR supplier. Before testing, the background was scanned from 4000 to 400 cm⁻¹ using a thin layer of KBr excluding the sample. Each sample was scanned 32 times, and the average was taken with a resolution of 4 cm⁻¹. Spectral analysis was conducted using Omnic 9.2 software (Thermo Scientific, MA, USA). The experiment was performed at room temperature [36].

2.11. Water absorption capacity, water solubility index, swelling power, and amylose content

The water absorption capacity (WAC) was determined using the method described by Cao et al. [37], with slight modifications. 2 g of corn starch (m₀) was added to a centrifuge tube (m₁) containing 20 ml of deionized water. The samples were stored at room temperature for 30 min and then centrifuged at 8000 rpm for 10 min. The supernatant was removed, and the centrifuge tube containing the precipitate was weighed (m₂). The WAC was calculated by following equation:

$$\mathrm{WAC}(\mathrm{g}/\mathrm{g})=\frac{m_2-m_1}{m_0}$$ (5)

The water solubility index (WSI) and swelling power (SP) were measured according to the description provided by Yan, Feng, Shi, Cui, and Liu [38]. Corn starch (2 g, W₀) and deionized water (20 ml) were mixed in a centrifuge tube. All the tubes were kept in a 90 °C water bath for 10 min and then cooled to room temperature. The samples were centrifuged at 8000 rpm for 10 min, and the supernatant was poured into an evaporation dish and weighed for precipitation (W_p). The dry weight of the supernatant (W_s) was determined by drying for 12 h at 110 °C. The WSI and SP were calculated using the following equations:

$$\mathrm{WSI}(\mathrm{g}/\mathrm{g})=\frac{W_s}{W_0}$$ (6)

$$\mathrm{SP}(\mathrm{g}/\mathrm{g})=\frac{W_p}{W_0-W_s}$$ (7)

The amylose content (AC) in starch samples was estimated using the iodine binding method, where 100 mg of starch sample was dispersed in 1 mL of 95 % ethanol and 9 mL of 0.1 N NaOH and boiled for 10 min. To 5 mL of the above solution, 1 mL of 1 N acetic acid and 2 mL of iodine solution were added and diluted to 100 mL. After incubating for 20 min, the absorbance of the solution was measured at 620 nm [39]. The amylose (%) was calculated using the following equation, where 3.06 is the conversion factor:

$$\%\mathrm{Amylose}=3.06\times \mathrm{Absorbance}\times 20$$ (8)

2.12. Statistical analysis

In this study, all assays were performed in at least three replicates, and the results were presented as means ± standard deviation (SD). The results were analyzed using SPSS 22.0 software (SPSS Inc., Chicago, USA). One-way analysis of variance (ANOVA) and least significant difference (LSD) analysis at a significance level of 0.05 were used to determine any significant differences between the groups.

3. Results and discussion

3.1. Characterization of plasma-activated water

The current and voltage waveforms, as shown in Fig. 2, depicted the current and voltage of a DBD operating at a frequency of 20 kHz. These waveforms represented the discharge behavior while treating distilled water over two applied voltage cycles. The voltage during each applied voltage cycle (0.1 ms) reached up to 27.38 kV. The average discharge power was directly calculated based on the current and voltage measurements reported in Fig. 2, amounting to 724.1 ± 14.28 W.

Fig. 2.

Voltage and current as a function of time.

EC is used to identify the concentration of reactive ions in water, and ORP serves as a measure of the overall oxidation capacity of a solution [40]. The physicochemical properties of each treated solution are shown in Table 1. Plasma activation led to a decrease in pH value and an increase in ORP and EC, resulting in the formation of reactive species such as H₂O₂, NO₃^–, and NO₂^–, consistent with previous studies' findings [[28], [41], [42]]. The reactivity of PAW was associated with the production of many high levels of ROS and RNS, EC values, NO₃^– and NO₂^– concentrations increased slightly after applying UL treatment (PUL), the same phenomenon Sun et al. [43] observed. These reactive species are key in altering starch properties [17].

Table 1.

Physicochemical properties of each treatment solution.

Samples	pH	ORP (mV)	EC (μS/cm)	H2O2 (μmol/L)	NO2– (mg/L)	NO3– (mg/L)
CK	/	/	/	/	/	/
SDW	6.18 ± 0.01a	217.33 ± 4.51b	1.39 ± 0.01c	ND	ND	ND
PAW	3.65 ± 0.12b	431.67 ± 6.03a	362.67 ± 2.51b	35.96 ± 1.38a	72.69 ± 1.94b	65.23 ± 1.69b
UL	6.13 ± 0.03a	225.67 ± 4.16b	2.81 ± 0.05c	ND	ND	ND
PUL	3.61 ± 0.1b	436.67 ± 4.51a	371.57 ± 3.07a	34.74 ± 1.86a	76.09 ± 1.99a	68.59 ± 1.4a

The results are expressed as the means ± SD. Values represented with different letters in the same column are significantly different (p < 0.05). ND = Not detected in the sample. CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved.

3.2. Color properties

The visual attributes of a product can be determined by color parameters L*, a*, and b*. The commercial value of starch is directly proportional to its color. The whiter the starch, the higher its commercial value [44]. The color characteristics of the corn starch are presented in Table 2. Compared to native starch (CK and SDW), after PAW treatment, the L* and b* values of the samples increased, while a* was lower, indicating higher purity of the starch, which could be attributed to plasma etching on the starch surface, which altered the surface roughness and, consequently, changed the light reflectivity of the treated starch granules [45]. Compared to native starch, the L* value for the ultrasonically treated starch was higher, whereas the a* and b* values were lower, which were consistent with the research findings of Kaul et al. [46]. The ΔE and WI increased after UL and PAW treatments, which could be related to the changes in crystalline structure induced by plasma and ultrasound modifications [15]. The UL treatment group exhibited the lowest chroma (C), which could be attributed to ultrasound promoting the dispersion and depolymerization of starch granules, thereby reducing granule aggregation. More dispersed granule may reduce the scattering of light, resulting in a decrease in chroma [47]. The C values of the PAW and PUL groups were higher than those of the UL group, indicating that PAW could enhance the chroma of the samples, making the surface colors more intense and saturated [48]. Overall, the changes in color parameters and WI could likely be attributed to the ultrasonic effects and the reactive species in PAW (primarily ROS and RNS), which oxidized the pigments into small fragments [49].

Table 2.

Color properties of native and modified corn starches.

Samples	Color parameters
	L*	a*	b*	ΔE	WI	C
CK	93.26 ± 0.08d	0.54 ± 0.02b	6.04 ± 0.03c	0	0	6.06 ± 0.04c
SDW	91.39 ± 0.06e	0.73 ± 0.02a	6.49 ± 0.03b	1.93 ± 0.02d	1.86 ± 0.03d	6.53 ± 0.03b
PAW	95.28 ± 0.04c	0.29 ± 0.02c	6.77 ± 0.05a	2.16 ± 0.03c	2.02 ± 0.04c	6.78 ± 0.05a
UL	97.23 ± 0.09b	0.16 ± 0.01d	4.16 ± 0.01e	4.41 ± 0.07b	3.97 ± 0.1b	4.16 ± 0.01e
PUL	98.45 ± 0.12a	0.05 ± 0.01e	5.16 ± 0.02d	5.29 ± 0.05a	5.19 ± 0.04a	5.16 ± 0.02d

The results are expressed as the means ± SD. Values represented with different letters in the same column are significantly different (p < 0.05). CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved. WI: whiteness index, C: chroma.

3.3. Morphology observation

The alteration in the structure of starch can be detected through microscopic analysis. The microstructure of corn was observed using SEM, CLSM, and OM. The SEM images of native and modified starches are shown in Fig. 3. The granules of native corn starch were irregular in shape and had a relatively smooth surface without any cracks or grooves. PAW treatment resulted in an etching effect, which, on one hand, increased the roughness of the granule surface structure. On the other hand, the reactive species in PAW might have reacted with the chemical substances on the starch surface and penetrated the interior of the granules, thereby intensifying the corrosion of the starch structure [50]. Ultrasonic treatment also eroded the surface of the starch granules, increasing the roughness of the starch surface. However, both single and dual modifications maintained the integrity of the starch granule structure. Typically, physical modification does not cause changes in the size and shape of starch granules, but it results in increased surface roughness and porosity. Sujka and Jamroz [19] also reported similar results, where ultrasonic treatment of starch led to the rapid formation and collapse of cavitation bubbles, generating intense shear forces, manifesting as small fissures and indentations on the granule surface. Under the continuous influence of ultrasound, the surface-damaged granules may reassemble through hydrogen bonding [51], with merged starch granules also observed in the PUL group. PAW treatment can create a rough structure and shallow pores on the surface of starch granules. Under the synergistic action of PAW and ultrasound, the porosity and cracks in the starch granules increase while maintaining the integrity of the granular structure.

Fig. 3.

Images of SEM (×500 magnification) (A), SEM (×2000 magnification) (B), CLSM (×200 magnification) (C), CLSM (×400 magnification) (D), and optical microscopy (×20 magnification) (E) for the native and modified corn starches.

The analysis of CLSM can detect the internal structure of starch granules as well as the presence of voids and cracks [52]. The arrangement of amorphous and crystalline regions in starch forms an annular structure. In this study, alternating light and dark growth rings were observed in all samples, along with unique pore structures and hilum centroids in the starch. The surfaces of native starch granules were smooth and crack-free. However, ultrasound and PAW treatment caused the starch granules to form cracks, with channels deepening from the granule center as the cracks increased. The annulus structure of the starch was lost due to the disruption of corn starch granules by PAW and UL. Ultrasound also affected the structure of corn starch granules, inducing cavities on their surfaces and more cracks or holes extending from the inside out, thereby providing channels that promote water diffusion into the granules, enhancing their water absorption capacity [53]. Hu et al. also obtained similar results, finding that ultrasound could cause starch granules to break apart due to high-frequency vibrations [54]. The reactive species of plasma penetrated the interiors of starch granules and interacted with starch molecules in the pores, thereby degrading starch chains and creating new pores due to the depolymerization of starch chains. The synergistic effect of PAW and UL made the porosity of starch granules most pronounced, consistent with the results of SEM. After PAW treatment, the fluorescence intensity around the channels of native starch granules decreased. This might have been due to changes in the crystalline and amorphous regions inside the starch caused by PAW treatment. It was observable that after ultrasonication, plasma treatment, and dual modification, the internal channels of the starch extended from the core to the periphery, indicating that all three modification methods altered the ordered structure of starch molecules. The Maltese cross of ultrasound and PAW-treated starch was observed to be insignificant by OM, indicating that the Maltese cross of starch was disrupted, which was consistent with the results of SEM and CLSM.

3.4. WAC, WSI, SP, and AC

The WAC and WSI determine the amount of water that can be retained by starch granules per unit weight and the degree of dissolution of the granules at a specific temperature. The WAC, WSI, and SP for native and modified starches are presented in Table 3. The results indicated that PAW treatment significantly increased the WAC and WSI values for corn starch while reducing the SP value, which was attributed to the bombardment of the starch granules by high-energy plasma particles, which loosened the surface structure of the granules and facilitated the ingress of water molecules. This process was accompanied by increased cracks and channels, enhancing the granule's WAC and WSI [55], which was consistent with the results of changes in the microstructure of starch granules. Additionally, PAW treatment might have led to breaking starch chains, reducing the strength of the three-dimensional structure formed by starch granules during the swelling process. Ultrasonic treatment significantly enhanced the WAC and WSI of starch due to the cavitation effect. The external structure of starch granules was damaged after ultrasonic treatment, making it easier for water molecules to penetrate the granules. This finding was consistent with the discoveries of Cao et al. [8]. Ultrasonic treatment increased the SP of starch. The enhancement of SP was due to the ultrasonic disruption of the native starch's molecular structure, thereby facilitating the ingress of water molecules, which disrupted the free hydroxyl groups (OH) of amylose and amylopectin [46]. The solubility of starch is related to the AC present or released from the starch, and ultrasonic treatment may lead to the leaching of amylose [8], this confirmed that a more significant amount of amylose was exposed after UL and PUL treatments, and such starch could be used to improve intestinal health and control blood sugar levels [56].

Table 3.

WAC, WSI, SP, AC, R_1047/1022, and R_1022/995 of native and modified corn starches.

Samples	WAC (g/g)	WSI (%)	SP (g/g)	AC (%)	R1047/1022	R1022/995
CK	1.81 ± 0.04c	0.48 ± 0.01c	5.3 ± 0.25b	25.61 ± 0.26c	1.67 ± 0.01c	1.01 ± 0.01c
SDW	1.82 ± 0.06c	0.49 ± 0.02c	5.28 ± 0.25b	25.58 ± 0.23c	1.68 ± 0.02c	1.02 ± 0.02c
PAW	2.11 ± 0.13b	1.11 ± 0.13b	5.15 ± 0.09b	17.19 ± 0.32d	1.73 ± 0.02b	0.96 ± 0.02d
UL	2.07 ± 0.06b	1.07 ± 0.11b	6.04 ± 0.08a	29.4 ± 0.24a	1.62 ± 0.02d	1.18 ± 0.02a
PUL	2.48 ± 0.08a	1.58 ± 0.23a	5.8 ± 0.17a	26.97 ± 0.08b	1.77 ± 0.02a	1.07 ± 0.02b

The results are expressed as the means ± SD. Values represented with different letters in the same column are significantly different (p < 0.05). CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved. WAC: water absorption capacity, WSI: water solubility index, SP: swelling power, AC: amylose content, R_1047/1022: the ratios of absorbance at 1047 cm⁻¹/1022 cm⁻¹ in the FTIR spectroscopy, R_1022/995: the ratios of absorbance at 1022 cm⁻¹/995 cm⁻¹ in the FTIR spectroscopy.

3.5. Fourier transform infrared (FTIR) spectroscopy analysis

Fig. 4A shows the infrared spectra of native and modified starches, and the results show that the positions of the characteristic absorption peaks of native and modified starches did not change. However, the amplitudes of the peaks were different. These observations indicate that PAW, ultrasound, and double modification neither change functional groups nor create new chemical bonds. These are all physical modification processes consistent with the previous findings of Davoudi et al. and Yılmaz et al. [[26], [57]]. The broadband near 3380 cm⁻¹ in the wavenumber range is associated with the stretching vibrations of intra- and intermolecular hydrogen bonds (OH), which contribute to the hydrophilicity of the substance. UL reduced the intensity of this spectral band, implying that the starch microstructure's ability to retain bound water was affected. The major absorption band observed near 2934 cm⁻¹ is a characteristic absorption band of polysaccharides, corresponding to the vibrations of the C-H bonds. The peak at 1649 cm⁻¹ is influenced by the bending vibrations of OH groups in water and the amorphous regions of starch [58]. The three peaks mentioned are typical absorption peaks for starch. The treatments with PAW and PUL increased the band intensity within the 3500 to 3200 cm⁻¹ range. This increase is attributed to the enhanced absorption by the hydroxyl groups present in the starch molecules [59]. The intensities of the peaks at 3380 and 2934 cm⁻¹ are higher in the starch samples treated with PAW and PUL than in native starch. These findings suggest that the quantity of hydrophilic groups in the starch structure increases after PAW treatment. The enhanced PV and WAC of the modified starch samples also confirm these observations.

Fig. 4.

FTIR pattern (A), DSC thermograms (B), and XRD (C) of corn starches with different pretreatments. CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved.

The absorption peak areas at 1047, 1022, and 995 cm⁻¹ correspond to the ordered crystalline regions, amorphous regions, and helical structures of starch, respectively [60]. To compare the samples more effectively, the absorbance ratios at these positions were utilized (Table 3). The increases in the intensity ratios of 1022/995 cm⁻¹ (R_1022/995) and 1047/1022 cm⁻¹ (R_1047/1022), respectively, indicate the disruption of the double helical structure and an increase in the short-range order of starch granules. After ultrasonic treatment, the samples showed a decrease in R_1047/1022, indicating that ultrasound disrupts the crystallinity of starch granules, which was consistent with the decrease in relative crystallinity in XRD. The increase in R_1047/1022 of the samples treated with PAW and PUL may be because PAW disrupts the molecular chains of starch, and the generated short-chain molecular chains are connected by hydrogen bonding to form a new ordered structure [61]. For the dual-modified starch samples, the PAW pre-treatment promoted the rearrangement of starch molecules and the formation of ordered structures, and the subsequent ultrasound treatment enhanced this change, resulting in higher R_1047/1022 than the single-treated starches. In the UL and PUL-treated samples, the R_1022/995 was 1.18 and 1.07, respectively, were significantly higher than those of native starch (p < 0.05), which indicated that the order of the double-helix structure in the starch granules was disturbed, and the ordered arrangement of starch molecules was reduced [62]. This was consistent with the findings of Shen et al. [18], who found that ultrasound-treated mung bean starch had a lower-ordered structure than native starch.

3.6. Thermal properties and XRD pattern analysis

The thermal properties of native and modified starches are presented in Fig. 4B and Table 4. All samples exhibited an endothermic peak, with the transition temperatures observed in DSC analysis representing the degree of perfection of the double-helical order. T_o represents the fusion of weaker crystalline structures, while T_c represents the fusion of stronger crystalline structures [63]. T_o, T_p, T_c and ΔH of native starch were 68.55 °C, 73.46 °C, 79.6 °C, and 11.22 J/g, respectively. The gelatinization parameters of starch treated with PAW were higher than those of native starch, which may be due to the oriented rearrangement and crystalline reorganization of starch molecular fragments under the action of PAW and PUL compared to native starches. This increased the orderliness of the amorphous regions of starch, resulting in more compressed and denser crystalline areas, thereby providing higher thermal stability against gelatinization for the granules. ΔH reflects the loss of molecular order (crystallinity and double helices), not just the crystalline structure [10]. A higher ΔH indicates a greater number and tighter packing of double helices. Ultrasonic irradiation disrupted the weak crystalline structures within the starch granules, making the remaining crystals more stable. Consequently, more energy is required to break the intermolecular bonds within the starch granules, increasing ΔH [10]. The FTIR study results, showing a decrease in R_1047/1022 for the UL group, also indicated the disruption of crystalline regions in starch. Additionally, ultrasonic irradiation disrupted the granular structure, facilitating water molecules' penetration into starch granules and the leaching out of amylose molecules from the starch interior. The leached amylose could partially gelatinize at high temperatures on the starch surface, thereby preventing the internal starch molecules from reacting with water, which increased ΔH [64]. The increase in ΔH indicates a higher thermal resistance of the starch granules. Therefore, corn starch treated with UL and PAW can withstand higher processing temperatures and be utilized as a heat-resistant absorbent.

Table 4.

The onset, peak, and conclusion temperature (T_o, T_p, and T_c, respectively) and gelatinization enthalpy (ΔH) of native and modified corn starches.

Samples	To (°C)	Tp (°C)	Tc (°C)	△H (J/g)
CK	68.55 ± 0.13d	73.46 ± 0.23c	79.6 ± 0.21d	11.22 ± 0.1c
SDW	68.3 ± 0.1d	73.45 ± 0.3c	79.74 ± 0.06d	11.15 ± 0.08c
PAW	69.69 ± 0.17c	75.48 ± 0.2b	81.44 ± 0.21b	12.58 ± 0.2b
UL	70.25 ± 0.12b	75.65 ± 0.06ab	80.52 ± 0.15c	12.53 ± 0.13b
PUL	72.49 ± 0.27a	75.89 ± 0.09a	83.66 ± 0.11a	13.13 ± 0.1a

Values represented with different letters in the same column are significantly different (p < 0.05). CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved.

XRD was utilized to investigate the effects of PAW and ultrasound on the amorphous and crystalline regions of starch. The principal diffraction peaks of all samples were observed at 2θ of 15.1, 17.3, 18, 20, and 23.1° (Fig. 4C), indicating that the crystalline form of corn starch is type A and the modification treatments did not affect the crystalline structure of the starch. Davoudi et al. [26] and Rahaman et al. [22] also reported that CP and ultrasound do not affect the crystallinity pattern of corn starch. However, the intensity of peaks for native and modified starches differed, leading to changes in the calculated relative crystallinity (RC). After ultrasound treatment, the RC of starch decreased. The instability of the starch's lamellar structure and the potential disruption of the double helix structure in the crystalline regions of amylose could be reasons for the reduced RC in the ultrasound-treated samples [22]. The RC of starch in the PAW and PUL treatment groups were significantly higher than those in the native starch and ultrasound treatment groups (p < 0.05), indicating that PAW can enhance the RC of starch. This could be due to the acidic components of PAW preferentially hydrolyzing the amorphous regions of starch, and the starch treated with PAW also exhibited cross-linking effects, where the aggregation of short chains or small fragments in the crystalline regions strengthened the starch crystal structure. Additionally, this behavior could also be attributed to the better crystalline orientation of the hydrolyzed starch [65]. The results of XRD were consistent with those of FTIR spectra and DSC.

3.7. Pasting properties of corn starch

The pasting curve of corn starch is presented in Fig. 5, with its pasting parameters listed in Table 5. It can be seen from Table 5 that there was a significant difference in the pasting properties (except for BV) between native corn starch and ultrasound-treated corn starch (p < 0.05). The cavitation effect produced by ultrasound severely damaged the internal and intermolecular hydrogen bonds of the starch chains, leading to a decrease in the content of free starch chains and branched starch. This made it easier for water molecules to penetrate the starch granules, accelerating the pasting process and lowering the pasting temperature [66]. Compared to the CK, ultrasound treatment significantly increased the starch's SV and FV (p < 0.05). Ultrasound disrupted the crystalline structure of the starch, causing the starch chains to break. Branched starch can fragment under high temperature and pressure, increasing the likelihood of contact and movement between starch molecules, thereby increasing the starch's viscosity [67]. A similar phenomenon was observed in rice starch by Wang et al. [68].

Fig. 5.

RVA curves of native and modified starches. The red line represents the trend of the heating temperature of starch with time in the determination of starch pasting properties. CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved.

Table 5.

Pasting properties of native and modified corn starches.

Samples	PT (°C)	PV (cP)	BV (cP)	SV (cP)	FV (cP)	TV (cP)
CK	74.2 ± 0.07c	3140.33 ± 12.01d	1326 ± 11.79ab	1645 ± 9.85b	3459.33 ± 12.01d	1814.33 ± 9.02c
SDW	74.5 ± 0.38c	3211 ± 10.54c	1279 ± 18.52bc	1621 ± 11c	3553 ± 12.77c	1932 ± 9.85b
PAW	77.6 ± 0.27a	3389 ± 20.78a	1263.33 ± 9.07c	1524 ± 10.58d	3639.33 ± 13.01b	2125.67 ± 14.64a
UL	72.99 ± 0.2d	3100 ± 11e	1341.67 ± 8.96a	1693.67 ± 5.03a	3752 ± 11.53a	1758.33 ± 14.57d
PUL	76.34 ± 0.19b	3347.33 ± 30.99b	1232 ± 51.51c	1645.67 ± 13.5b	3771.33 ± 2.52a	2115.33 ± 21.94a

Different letters in each column indicate significant differences (p < 0.05). CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved. PT: paste temperature, PV: peak viscosity, BV: breakdown viscosity, SV: setback viscosity, FV: final viscosity, TV: trough viscosity.

Compared to native starch (CK and SDW), the PT of corn starch treated with PAW and PUL was higher, indicating its stronger resistance to swelling. PV is defined as the maximum viscosity measured during the heating of starch in the RVA, reflecting the starch's water-binding capacity [69], The cross-linking of starch molecules induced by plasma oxidation might have resulted in a higher PV. BV represents the thermal resistance of swollen starch granules in the paste during shearing and heating, meaning that a high BV indicates the granules' disintegration and their low resistance to shear forces [70]. The results indicated that the BV for the PAW and PUL treatment groups was lower than that for the native corn starch and the UL group corn starch, possibly due to the enhanced strength of the starch granule structure during the modification process [71]. These results were consistent with XRD findings, which showed a significant increase in starch crystallinity after PAW and PUL treatments. The reduction in BV suggests higher stability during the shearing and heating processes [70]. Therefore, PAW-treated starch could be used as a thermally stabilizing compound in starch-based dairy desserts and canned products [26]. SV indicates a tendency for amylose chains to retrograde and recrystallize [72]. This parameter was higher in the UL and PUL samples than in the native starch. High SV suggests an increased leakage of amylose from the granules, which then reassociates during cooling to form helical structures [72]. After PAW treatment, the SV of the starch paste decreased. Considering the enhanced resistance to retrogradation in starch after PAW treatment, this modified starch can be used to prevent stalling. The disruption of hydrogen bonds during starch modification facilitates the incorporation of water molecules, increasing viscosity [67]. A possible explanation is that reactive species in the plasma cause hydrogen bond breakage or weakening, resulting in starch chain depolymerization or a change in the ratio of starch to amylose [59]. Conversely, there is a report that after using a different type of plasma, plasma treatment reduced the viscosity of corn starch paste [15]. Therefore, the impact of PAW treatment on the pasting properties of starch is related to both the variety of starch and the plasma treatment conditions. All treatment methods increased the FV, which could be due to ultrasound and cold plasma releasing linear starch fragments, causing reactions in the starch paste upon cooling [73]. High-viscosity starch pastes help improve food stability, reduce water precipitation, and maintain the quality and taste of food products. Starch with a high FV is also more likely to form a gel when cooled, which is essential for producing jellies, puddings, candies, and other food products [74].

3.8. Gel texture properties

The textural properties of the gel are related to the network structure and strength of the starch macromolecules involved in its formation and reflect the quality of the starch to a certain extent. The hardness and elasticity of various modified gels are shown in Fig. 6. Ultrasound treatment reduced the hardness of the gel and improved its elasticity, making it more suitable for consumption by the elderly, children, or individuals with swallowing difficulties. This is primarily due to ultrasound's effect on disrupting and reorganizing the amylose and branched starch in the starch, leading to changes in the textural properties of the gel [75]. PAW treatment increased the elasticity and hardness of the gel, as plasma treatment can enhance intermolecular or intramolecular cross-linking in starch molecules [76]. This cross-linking can increase the interconnectivity between starch molecules, allowing the gel network to more easily return to its original state after being subjected to an external force, thereby enhancing the structural stability and elasticity of the gel [77]. The increase in hardness might be due to the reactive species in PAW inducing cross-linking reactions in starch, leading to the rearrangement of starch chains [55]. PAW may also cause the depolymerization of starch granules, and the small molecules produced by the depolymerization reaction can form numerous bonds, thereby increasing the hardness of the gel [78].

Fig. 6.

Gel texture analysis of native and modified starches. Different uppercase and lowercase letters indicate significant differences among the different gel samples (p < 0.05). CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved.

3.9. Principal component analysis (PCA) and partial least squares discrimination analysis (PLS-DA)

PCA was employed to analyze the structure and various properties of five starch samples, as shown in Fig. 7A, where PC1 and PC2 explained 51.3 % and 38.9 % of the total variance, respectively, accounting for 90.2 %. Native starch and PAW-treated starch were differentiated through the positive and negative quadrants of PC2. The PUL group was positioned diagonally opposite to native starch, while UL was diagonally opposite to PAW. Therefore, PCA could identify native and modified starches to a certain extent. Additionally, the pasting properties (PT, PV, SV, FV, and TV), hardness, R_1047/1022, and RC, along with thermal properties (T_o, T_p, T_c, and ΔH), WAC, and WSI, were closely positioned for the PUL group starches in the score plot, indicating significant correlations. The color parameters L*, WI, ΔE, b*, and C, along with the PAW-treated samples, were proximal in the score plot, suggesting a high correlation. The AC, R_1022/995, SP, and PAW-treated samples were also closely located in the score plot, indicating significant correlations. PCA established strong relationships between the primary characteristics of starch and exhibited some clustering of native and modified starches.

Fig. 7.

PCA biplot summarizing the relationship between native and modified starch granules and their microstructures and key properties (A) PLS-DA analysis score plot (B) and associated VIP scores obtained from the PLS-DA model (C). CK: corn starch without any treatment, SDW: corn starch mixed with distilled water, PAW: corn starch mixed with plasma-activated water, UL: corn starch mixed with distilled water then ultrasound treatment, PUL: corn starch mixed with plasma-activated water then ultrasound treatment. All treated samples were dried overnight in an oven at 45 °C, ground, and sieved.

The discriminatory performance of PCA for components with insignificant between-group differences still needs to be improved. The PLS-DA score plots showed a better model fit (more significant intergroup differences) than PCA. In order to gain a deeper understanding of the characterization between native and modified starches, supervised analyses using PLS-DA effectively discriminated between groups based on measurements, with PC1 and PC2 accounting for 63.7 % and 29.4 %, respectively (Fig. 7B). In order to obtain more information about the differences in measurement indices between the five groups, the significant variable in prediction (VIP) scores was evaluated (VIP value > 1 is usually considered significant measurement indices for the model under study). Fifteen compounds were screened and ranked according to their VIP values (Fig. 7C). The results showed that five pasting parameters had VIP values greater than 1: FV, BV, TV, SV, and PV. This indicated that this pasting property plays a vital role in modifying corn starch and may affect various qualities of starch to some extent.

4. Conclusion

The study investigated the effects of two environmentally friendly technologies, ultrasound and PAW, on structural, thermal, physicochemical, functional, and pasting properties of corn starch. The cavitation effect, mechanical effect, and transient local high temperature of ultrasound, along with the acidic environment, hydrogen peroxide, nitrate, nitrite, and other reactive components of PAW, played significant roles in modifying corn starch. Both ultrasound and plasma disrupted the amorphous and weak crystalline structures of the starch. Compared to single treatments, dual treatment enhanced the modification effects on starch. The synergistic action of ultrasound and plasma-activated water (PAW) treatment increased the starch's gelatinization temperature and enthalpy, with the rise in ΔH indicating greater thermal resistance of the starch granules. The dual-modified starch showed increased relative crystallinity and gel strength without altering the crystal type. The whiteness index, water absorption capacity, water solubility index, and swelling power were improved compared to native and singly modified starches, and the starch's lamellar and double helical structures were disrupted. This study provides theoretical support for applying ultrasound-PAW dual-modified starch and offers a new pathway to understanding the mechanisms of physical starch modification. Further research is needed to fully understand the changes in starch-PAW-UL interactions and promote the application of modified starch in the industrial production of food ingredients, assessing its functionality in actual food formulations and its interaction with other food components.

CRediT authorship contribution statement

Yongxuan Zuo: Writing – original draft, Investigation, Formal analysis, Conceptualization. Fanglei Zou: Writing – review & editing, Writing – original draft, Visualization, Methodology, Conceptualization. Miao Yang: Methodology, Investigation. Guangfei Xu: Investigation. Junhua Wu: Methodology. Liangju Wang: Writing – review & editing, Conceptualization. Hongying Wang: Supervision, Resources, Project administration, Funding acquisition.

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.