Planetary Ball-Milling on Cornstarch

Effect of Planetary Ball-Milling on Cornstarch

Abstract

This paper investigates the impact of mechanical modification via planetary ball-milling on the multi-scale structures and pasting properties of waxy and high-amylose cornstarches.
High-amylose cornstarch: Demonstrated high resistance to structural and property changes during ball-milling due to thicker semi-crystalline lamellae, larger crystalline amylopectin lamellae, thinner amorphous amylopectin lamellae, and a more rigid amylose amorphous background region.
Waxy cornstarch: Showed distinct changes, including reduced pasting temperature and paste viscosity, increased pasting stability, and reduced retrogradation.
Suggests planetary ball-milling is a potential physical method for obtaining starch products with relatively low viscosity at high concentrations and enhanced pasting stability.

1. Introduction

Starch is a natural polysaccharide widely used in food and non-food products.
Starch structure determines its properties, making it crucial to achieve desirable structures for specific applications.
Native starch's complex structure limits its application, necessitating techniques to improve its functional and physicochemical properties like pasting and gelatinization behaviors.
Physical techniques for starch modification (heat-moisture, ultrasound, microwave, high-pressure, and ball-milling) are gaining attention due to increased safety and reduced waste generation.
Ball-milling is an eco-friendly and cost-effective physical technique that can regulate starch structure and modify physicochemical properties of starch and cereal flour through friction, collision, and shear from grinding balls and the container wall.
Ball-milling can significantly modify granule morphology, size distribution, crystallinity, molecular weight, and amylose/amylopectin ratio of starch, resulting in changes in solubility, digestibility, pasting, and rheological properties.
Ball-milling shows potential as a physical approach to modulate starch structure and functionalities for expanding its applications.
Starch is a mixture of amylose (10-30%, mostly linear 1,4-α-D-glucan with a few long branches) and amylopectin (70-90%, mainly 1,4-α-D-glucan with many 1,6-α linkages at branch points).
High-amylose starch can have up to 85% amylose, while waxy starch can contain 100% amylopectin after genetic modification.
These biopolymers form starch's hierarchical structure organized on multi-length scales: whole granule, growth rings, semi-crystalline lamellae, and crystalline structure.
Waxy, regular, and high-amylose starches exhibit prominent differences in hierarchical structure due to different amylose/amylopectin ratios.
Waxy and regular starches have A-type crystallites, while high-amylose starch predominantly displays a B-type crystalline structure.
Starch with higher amylose content is less susceptible to physicochemical treatments despite lower crystallinity.
Understanding the effects of ball-milling on the hierarchical structure and properties of starches with different amylose/amylopectin ratios is vital for improving functional properties.
Planetary ball-milling can cause more apparent alterations to starch characteristics compared to conventional methods, making it more suitable for starch modification; it can reduce starch crystallinity and double-helices.
The changes induced by planetary ball-milling in starch multi-scale structures and functionalities such as pasting properties have not been well understood, especially when starches with different amylose/amylopectin ratios are involved.
This study selects waxy and high-amylose (Gelose 80, or G80) cornstarches treated by a planetary ball mill for varying times to explore changes in granule morphology, granule size distribution, semi-crystalline lamellae, crystalline structure, molecular structure, and pasting properties.

2. Materials and methods

Materials:
- Waxy cornstarch (0/100 amylose/amylopectin ratio) from Lihua Starch Industry Co., Ltd. (Qinhuangdao, China).
- High-amylose cornstarch, Gelose 80 (G80) (80/20 amylose/amylopectin ratio) supplied by Penford (Australia).
- Moisture content (MC) of each sample (about 10%) determined using a moisture analyzer (MA35, Sartorius Stedim Biotech GmbH, Germany).
- Anhydrous ethanol in reagent grade from Nanjing Chemical Reagents Co., Ltd. (Nanjing, China).
Planetary ball-milling treatment:
- A QM-BP planetary ball mill (Nanda, Nanjing, China) with four ceramic milling cylinders (100 mL) was used.
- About 15 g of starch and zirconia balls (a mixture of balls with diameters of 2, 5, and 10 mm) with a weight three times that of starch were placed into each ceramic container, filling about 1/3 capacity of the container, followed by the addition of 12 mL of anhydrous ethanol.
- The cylindrical container was tumbled at a rotation speed of 1032 rpm (the ratio of rotation/revolution speed, 2/1) for 4, 8, 15, or 20 h.
- After treatment, the sample was collected after removing the balls and ethanol (by evaporation) and then sealed for further analyses.
- The code typically as “Waxy-0h” is used; “Waxy” represents the type of cornstarch, and “0h” indicates the ball-milling treatment time.
Scanning electron microscopy (SEM):
- Granule morphology was observed using an EVO18 scanning electron microscope (ZEISS, Germany), operated at 10.0 kV. All the samples were coated with a thin gold film before the microscopic observation.
Laser diffraction analysis:
- Granule size distribution was analyzed by a Malvern Mastersizer 2000 laser-diffraction analyzer (Version 5.22, Malvern, UK) using a 1000 mL flow-through reservoir.
- Each ball-milled starch sample was added to the reservoir and fully dispersed in anhydrous ethanol until an obscuration value between 12% and 17% was achieved. The pump speed was set at 2050 r/min.
- The refractive index of the starch samples and the dispersing reagent ethanol was 1.54 and 1.36, respectively.
- Volume size distribution between 0.10 and $104.71 µm$ was recorded for all samples. All the results are reported as the averages of three replicates.
Small-angle X-ray scattering (SAXS):
- SAXS experiments were performed on a SAXSess small-angle X-ray scattering system (Anton-Paar, Austria) equipped with a PW3830 X-ray generator (PANalytical), operated at 50 mA and 40 kV, using Cu-Kα radiation with a wavelength of $0.1542 nm$ as the X-ray source.
- The samples (ca. 60% MC) used for the SAXS measurement were prepared by premixing the starches with added water in glass vials and were equilibrated at 20 °C for 24 h before the analysis. Each sample was placed in a paste sample cell and was exposed at the incident X-ray monochromatic beam for 5 min.
- The data, recorded using an image plate, were collected by the IP Reader software with a PerkinElmer storage phosphor system. All data were normalized, and the background intensity and smeared intensity were removed using the SAXSquant 3.0 software for further analysis.
Light microscopy:
- Both ordinary and polarized light micrographs were recorded using a light microscope (Axioskop 40 Pol/40APol, ZEISS, Oberkochen, Germany) equipped with a camera (PowerShot G5, Canon, Tokyo, Japan). The magnification was set at 500 ( $50 \times 10$ ). Each sample was dispersed as 10 mg of the starch in 1 mL of distilled water in a glass vial. Then, a drop of the starch suspension was transferred onto a slide, covered by a cover slip.
X-ray diffraction (XRD):
- X-ray diffraction patterns of the starch samples were measured using an Xpert PRO diffractometer (PANalytical, Netherlands), operated at 40 mA and 40 kV, using Cu-Kα radiation with a wavelength of $0.1542 nm$ as the X-ray source. The diffraction angle ( $2\theta$ ) scanning was from 5° to 40° with a scanning speed of 10°/min and a scanning step of 0.033°. The MC of each sample was about 10%.
Fourier transform Raman (FT-Raman) spectrometry:
- FT-Raman spectra were recorded on a Nicolet iS50 instrument (ThermoFisher, USA) with a near-infrared YAG laser with a wavelength of $1064 nm$ . The laser was focused on the sample at a spot of ca. 1 mm in diameter with a power of 400 mW. 1024 scans were used for each spectrum in the wavenumber range of 4000 to $30 cm^{-1}$ with a resolution of $8 cm^{-1}$ .
- The direct comparison of the spectrum intensities of different samples was reliable, as the layer thickness and spot laser size were practically identical for all samples.
Brabender viscoamylograph profiles:
- Pasting properties were analyzed on a BrabenderMicro Viscoamylograph machine (Brabender OHG, Germany). 100 g of 6.0% (w/w) starch suspensions were stirred at a paddle speed of 250 rpm. Each starch suspension was heated from 30 to 95 °C at $1.5 °C/min$ , held at 95 °C for 30 min, cooled from 95 to 50 °C at $1.5 °C/min$ , and held at 50 °C for 30 min. The data were processed and obtained using the Viscograph Data Correlation software.
Statistical analysis:
- The experiments were conducted in triple (n=3), and data were analyzed with SPSS 20.0 statistical software. Analysis of variance (ANOVA), followed by the Duncan’s multiple-range test, set the significance level at p < 0.05.

3. Results and discussion

3.1. Granule morphology
- SEM micrographs (Fig. 1) show that waxy cornstarches displayed more pronounced changes in granule morphology after planetary ball-milling compared to G80 cornstarch.
- Native waxy cornstarch consisted of large oval and polyhedral granules with slightly rough surfaces. As time increased to 15 h, granules were gradually broken into small fragments with rougher surfaces due to the mechanochemical effect of planetary ball-milling.
- Further increase up to 20 h did not substantially change the granule morphology of waxy cornstarch, suggesting a “grinding equilibration” at a ball-milling treatment time of about 15 h.
- Treatment for up to 20 h caused negligible changes to the granule morphology of G80 cornstarch, with only a slight increase in surface roughness, indicating greater resistance to mechanical force.
- This aligns with the fact that starch with higher amylose content often has stronger resistance to various physicochemical treatments.
3.2. Granule size distribution
- Evolutions of granule volume size distributions (Fig. 2a, b) and related parameters (Table 1) show that both cornstarches presented a decrease in granule size after treatment, indicated by a shift of the size distribution profile towards a smaller particle size range.
- This trend was more obvious when the amylopectin content was higher (waxy cornstarch), consistent with SEM results.
- d(0.5) (diameter value less than which 50% of the overall granules have) of waxy cornstarch reduced from $23.7 µm$ to $4.6 µm$ , while G80 cornstarch showed a less significant decrease from $12.5 µm$ to $8.3 µm$ .
- Grinding equilibration was also observed for G80 cornstarch at about 4 h because the granule size distributions are very similar for the G80 cornstarch planetary ball-milling treated between 4h and 20h.
- Waxy cornstarch showed a slight decrease in the proportion of fragments with the particle size between 0 and $1 µm$ , suggesting re-aggregation (agglomeration) of small fragments during planetary ball-milling treatment.
- Larger waxy cornstarch granules can be easier to damage by planetary ball-milling treatment.
- Since G80 cornstarch after the treatment for 4 h had larger granules than that of waxy cornstarch counterpart, the larger granule size should not be a predominant determinant for reducing the resistance of starch to planetary ball-milling treatment.
3.3. Lamellar structure
- Semi-crystalline lamellae are studied by SAXS.
- Using Woolf-Bragg’s equation, $d = 2\pi/q$ , the semi-crystalline lamellae average thickness (d) can be calculated using the q position of a scattering peak at around $0.6 nm^{-1}$ .
- Native waxy and G80 cornstarches possessed a scattering peak at 0.6270 and $0.5929 nm^{-1}$ , corresponding to a d value of 10.01 and $10.59 nm$ , respectively (Table 1), indicating that the semi-crystalline lamellae of the native waxy cornstarch were thinner than that of G80 cornstarch.
- After treatment, the semi-crystalline lamellae thickness of waxy cornstarch gradually decreased from 10.01 to $9.53 nm$ , and even disappeared when the time was $\geq 15 h$ . In contrast, very small changes occurred to the thickness of semi-crystalline lamellae of G80 cornstarch after the treatment.
- Planetary ball-milling treatment decreased the visibility of peak at around $0.6 nm^{-1}$ , suggesting structural disorganization with a reduction in the perfection and in ordering degree of the semi-crystalline structure.
- Semi-crystalline of waxy cornstarch underwent a more prominent destruction than that of G80 cornstarch, although the native waxy cornstarch had a more visible SAXS peak at around $0.6 nm^{-1}$ .
- Other two parameters of a theoretical model for the semi-crystalline lamellae in the starch granule obtained from the SAXS data:
 - $\Delta\rho = \rhoc - \rhoa$ (where $\rhoc$ and $\rhoa$ are the electron densities of the crystalline and amorphous regions in the semi-crystalline lamellae, respectively), the difference in electron density between the crystalline and amorphous lamellae.
 - $\Delta\rhou = \rhou - \rhoa$ (where $\rhou$ is the electron density of the amorphous background), the difference in election density between the amorphous lamellae and the amorphous background.
- A decrease in the definition of the peak (around $0.6 nm^{-1}$ ) was observed for waxy cornstarch with the time increasing up to 8 h, together with an increase in the scattering intensity at low q values. This indicates a decreased $\Delta\rho$ and an increased $\Delta\rho_u$ resulting from the greatest destruction to the crystalline lamellae, the intermediate destruction to the amorphous lamellae, and the weakest destruction to the amorphous background.
- Planetary ball-milling treatment for even longer times (15 and 20 h) would make the semi-crystalline lamellae of waxy cornstarch undetectable, presumably due to a higher degree of disorganization in the semi-crystalline lamellae.
- G80 cornstarch after the planetary ball-milling treatment presented moderate changes in the SAXS patterns, i.e., an increase in the overall intensity at low q values, but a decrease in the definition of the scattering peak (which became broader). This indicates that $\Delta\rho$ and $\Delta\rho_u$ of G80 cornstarch could be simultaneously increased by planetary ball-milling, due to the greatest destruction to the amorphous lamellae, the intermediate destruction to the amorphous background materials, and the weakest destruction to the crystalline lamellae.
- Waxy cornstarch contains only amylopectin with more short A-chains (DP 6–12), and thus has thinner crystalline amylopectin lamellae but thicker amorphous amylopectin lamellae.
- G80 cornstarch has ca. 80% amylose which presents in the amorphous background region, imparting a 'harder' structure to such region, while ca. 20% amylopectin in G80 cornstarch forms larger crystalline amylopectin lamellae and thinner amorphous amylopectin lamellae.
- With thicker semi-crystalline lamellae, larger crystalline amylopectin lamellae, thinner amorphous amylopectin lamellae and a larger amount of amylose amorphous background region with structural rigidity, G80 cornstarch showed higher resistance to the mechanical disruption during the planetary ball-milling treatment.
3.4. Crystalline structure
- Starch granule is a semi-crystalline system consisting of crystalline and amorphous regions, and when it is under polarized light, a polarization cross (birefringence) can be seen.
- Moreover, as the intensity of birefringence is related to the granule size, degree of crystallinity and microcrystalline orientation, the destruction to the crystalline structure of waxy cornstarch by planetary ball-milling could conceivably result in reduced birefringence intensity, but for the G80 cornstarch there were negligible changes in the birefringence.
- This indicates that planetary ball-milling treatment could hardly destruct the crystalline lamellae of G80 cornstarch.
- The crystalline parts of starch showed sharp peaks but the rest amorphous parts presented dispersive patterns.
- The native waxy cornstarch displayed a typical A-type crystalline structure with main diffraction peaks at $\,2\theta$ of around 15°, 17°, 18° and 23°, whereas native G80 cornstarch displayed a B-type crystalline structure.
- After the treatment for different times, a substantial reduction in the intensities of A-type crystalline peaks occurred for waxy cornstarch, whereas G80 cornstarch only showed slightly changed XRD patterns.
- A-type crystalline starch is likely to contain the $\,\alpha$ -1,6 branch linkages, which are more scattered and are located within both the crystalline region and the amorphous region, while most of the branch linkages in the B-type crystalline starch are clustered in the amorphous region.
- The scattered branch points located inside the crystalline region leads to “weak points” in the A-type crystalline starch granules.
- A-type crystalline lamellae region was easier to damage.
- SAXS test shows a decreased $\,\Delta\rho$ and an increased $\,\Delta\rho_u$ resulting from the greatest destruction to the crystalline lamellae for waxy cornstarch.
- The A-type crystalline structure has a monoclinic crystal unit with 8 inter-helical water molecules, whereas the B-type crystalline structure has a more open packing of helices with 36 inter-helical water molecules in each hexagonal crystal unit.
- During planetary ball-milling, the larger amount of inter-helical water in the B-type crystallites could contribute to more hydrogen bonds and a better hydrogen bonding network, making B-type crystalline lamellae of G80 having greater resistance to the mechanical force during planetary ball-milling, which has been observed by the SAXS test.
- The amylose molecules could act as the backbones of the aggregation structures to provide resistance to the physical treatment of heat-moisture.
- B-type crystalline starch has a higher ordering degree of surface structure, acting as a factor to enhance the resistance to planetary ball-milling for the B-type G80 cornstarch.
- Planetary ball-milling would more powerfully alter the organization of waxy cornstarch crystallites, and thus a more prominent amorphisation could be observed for waxy cornstarch.
3.5. Molecular chain characteristics
- FT-Raman technique is used for investigating the morphology and structures of polymers.
- The FT-Raman spectra shows a similar pattern for native waxy and G80 cornstarches. Specifically:
 - The band at $2910 cm^{-1}$ was attributed to the O-H and C-H stretching vibrations.
 - The band $480 cm^{-1}$ was ascribed to the skeletal modes involving the (C-O-C) ring and $\,\delta$ (C-C-O).
 - The peak at $860 cm^{-1}$ originates from $\,V_s$ (C1-O-C5) ring mode and C1-H bending of $\,\alpha$ -configuration.
 - The peak at $940 cm^{-1}$ assigned to $\,V_s$ (C1-O-C4’) of $\,\alpha$ -1,4-glycosidic linkage.
 - The peak at $1390 cm^{-1}$ due to $\,\delta$ (C-OH), C-H bending and $\,CH2$ scissoring vibrations, and the peak at $1460 cm^{-1}$ resulting from $\,\delta(CH2)$ twisting and C-H bending.
- After planetary ball-milling, the FT-Raman intensity for waxy cornstarch in the range of 960 to $800 cm^{-1}$ was reduced gradually as the time increased.
- The breakage of waxy cornstarch glycosidic linkage occurred, since the peak intensity at $940 cm^{-1}$ $ related to C1 and C4 was reduced.
- The glucose units of waxy cornstarch were substantially degraded into short fragments by the planetary ball-milling treatment, as indicated by the shift and decrease of the peak at $860 cm^{-1}$ .
- G80 cornstarch showed negligible changes in the FT-Roman spectra, indicating no apparent alteration to its molecular chains.
- Compared with waxy cornstarch, G80 cornstarch possessed more robust supramolecular structures (semi-crystalline lamellae, and crystalline structure, etc.), which had greater resistance to the mechanical force of planetary ball-milling. This robustness could minimize the destruction to the molecular chains.
3.6. Pasting properties
- The typical pasting profile representing the gelatinization process cannot be observed for native G80 cornstarch since the highest temperature (95 °C) during the measurement is insufficient for the melting of crystalline structure of native G80 cornstarch.
- The G80 cornstarch sample after the treatment for 20 h did not present a gelatinization-like pasting profile, consistent with the negligible changes in the hierarchical structure of G80 cornstarch after planetary ball-milling treatment.
- For waxy cornstarch, a decrease in the pasting parameters was observed after the treatment for a prolonged time. This was caused by the prominent structural destruction to waxy cornstarch by the treatment.
- Not only could the decreased granule size (i.e., increased specific surface area) with broken surface promote the water permeability into the granules, but also the disorganization in the lamellae and crystallites could weaken the thermal stability of the supramolecular structures.
- The modified waxy cornstarch samples displayed weaker resistance to the swelling and rupture while being heated in water, resulting in a reduced pasting temperature ( $T_p$ ) as shown in Table 3.
- Despite the less ordered degree of the supramolecular structure could enhance the granule swelling, the swollen and/or broken granules had a fragile surface, which could decrease the resistance to shear, and therefore reduce their swelling degree.
- A reduction in the maximum viscosity ( $\,\eta_{pk}$ ) occurred for the treated waxy cornstarch samples.
- The paste viscosity ( $\,\eta{sc}$ ) at the start of cooling showed less significant difference without and with planetary ball-milling, although the treatment induced breakage of the molecular chains which could decrease the $\,\eta{sc}$ value.
- The modified waxy cornstarch samples displayed a smaller viscosity breakdown ( $\eta{bd}, \eta{pk} - \eta_{sc}$ ) than that of the native sample, indicating their higher paste stability at 95 °C.
- The treated granules displayed a reduced swelling degree and were less intact, made them less easy to be ruptured during the pasting process from $\,\eta{pk}$ to $\,\eta{sc}$ , and resulted in the reduced viscosity breakdown.
- The degraded molecular chains of waxy cornstarch were less easy for rearrangement.
- The treated waxy cornstarch samples had a lower tendency to retrogradation and higher paste stability during cooling as demonstrated by the reduced $\,\eta{sb} (\eta{ec} - \eta_{sc})$ (Table 3).
- The reduced tendency to retrogradation could also contribute to a more stable viscosity during paste holding at 50 °C.
- Grinding equilibration could be achieved at 15 h, and further treatment by planetary ball-milling would not result in any further change to the pasting parameters for waxy cornstarch.

4. Conclusion

The variation in the starch supramolecular structures by the amylose/amylopectin ratio could result in a different degree of resistance of starch to the planetary ball-milling treatment.
Compared with G80 cornstarch, the waxy cornstarch was more susceptible to planetary ball-milling, as shown by more significant changes in the multi-scale structures and pasting properties. This could result from the less robust supramolecular structures of waxy cornstarch.
This enables planetary ball-milling to be a potential physical technique to produce starch products with desired pasting behaviors, for expanding the applications of starch in foods and non-food products.
The data from the current work provides not only better understanding of the effects of planetary ball-milling on the characteristics of starches with different amylose/amylopectin ratios, but also fundamental knowledge in design of planetary ball-milling processes for more accurate physical modification of starches in the future.