Insight into the formation mechanism of lotus seed starch-lecithin complexes by dynamic high-pressure homogenization

Food Chemistry 315 (2020) 126245

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Insight into the formation mechanism of lotus seed starch-lecithin complexes by dynamic high-pressure homogenization ⁎

Yixin Zhenga,b, Zebin Guoa, Baodong Zhenga,b,c, Shaoxiao Zenga,b,c, , Hongliang Zenga,b,c,

T ⁎

a

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China Fujian Provincial Key Laboratory of Quality Science and Processing Technology in Special Starch, Fujian Agriculture and Forestry University, Fuzhou 350002, China c China-Ireland International Cooperation Centre for Food Material Science and Structure Design, Fujian Agriculture and Forestry University, Fuzhou 350002, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Lotus seed starch Lecithin Dynamic high-pressure homogenization Structural property Visual correlation analysis Formation mechanism

Our objective was to investigate the correlation between processing conditions and structural properties of lotus seed starch-lecithin complexes by dynamic high-pressure homogenization and explore the formation mechanism. The complexes formed with 5% lecithin at 90 MPa had the highest complex index among samples, thus protecting the integrity of the particles. The complexes inhibited the degradation of amylopectin and retrogradation of amylose, and displayed different V6II-, V6I- and A-type crystalline patterns. Additionally, the double helix structure was enhanced with increasing pressure, and the addition of lecithin contributed to the formation of single-helix amylose-lecithin complexes. These complexes prevented the single helix structure of starch to further form double helix structures, as demonstrated by visual correlation analysis. Moreover, a formation mechanism was established, and lotus seed starch-lecithin complexes with V6I-type crystalline were formed under appropriate conditions, but a homogenization pressure either too low or too high was not conducive to complex formation.

1. Introduction Lotus, a perennial herbaceous aquatic plant of the genus Nelumbo, is widely grown in Asia (Nelumbo nucifera Gaertn.) and the Americas (Nelumbo lutea) (Sharma, Gautam, Adhikari, & Karki, 2017). Chinese lotus (Nelumbo nucifera Gaertn.) can be divided into three major cultivation types depending on cultivation purposes, namely rhizome lotus, seed lotus and flower lotus (Zhu, 2017; Zhu, Liu, & Guo, 2016). Lotus seed, the mature seed from the seed lotus, is an important commercial crop in China. Lotus seed is often processed into lotus juice, canned lotus seed, lotus porridge and other products, because of its high nutritional value and pharmacological activity (Zhang et al., 2015). Deterioration and precipitation of the products often occurs, because lotus seed starch contains a high level of amylose (~40%, w/w), which may contribute to the retrogradation of lotus seed starch, thus resulting in precipitation during processing or storage (Zeng et al., 2017). The taste, color, morphology and storage stability of starchy foods can be affected by starch retrogradation (Kumar, Brennan, Zheng, & Brennan, 2018), thus limiting the use of lotus seed and lotus seed starch (Zeng, Chen et al., 2018; Zeng, Zheng et al., 2018). At present, starch modification technology, such as chemical modification, physical modification, enzymatic modification and composite

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modification, is usually used to improve starch structure and expand its range of uses (Haq et al., 2019; Lin, Liang, Zhong, Ye, & Singh, 2018). Physical modification methods have favorable characteristics such as safety and ease of operation. These methods are utilized to prepare starch-lipid complexes to improve the structural and processing properties of starch (Guo et al., 2018). The complexes formed from a combination of starch and lauric acid through water bath heating show significantly increased starch gel strength and inhibited gelatinization expansion of the starch (Chang, He, & Huang, 2013). The starch-stearic acid complexes under high-temperature cooking effectively delay the aging of starchy food and enable good storage stability of starch in aqueous media (Reddy, Choi, Lee, & Lim, 2018). V-type structure complexes formed by wheat starch with A-type crystal and lipids showed an improved crystalline region of starch crystals and increased starch crystallinity (Niu et al., 2019). Lecithin is an amphiphilic molecule riching in unsaturated fatty acid that imparts good emulsification and surface dispersion to starch (Züge, Haminiuk, Maciel, Silveira, & Scheer, 2013). Lecithin could form a complex with the de-branched starch spiral cavity through hydrophobic interaction, which significantly enhance the water retention and dispersion property of native starch (Cheng, Luo, Li, & Fu, 2015). However, there are few studies to discuss the formation mechanism of lotus seed starch-lecithin

Corresponding authors at: College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China. E-mail addresses: [email protected] (S. Zeng), [email protected] (H. Zeng).

https://doi.org/10.1016/j.foodchem.2020.126245 Received 19 April 2019; Received in revised form 13 December 2019; Accepted 16 January 2020 Available online 21 January 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.

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pulverized and passed through a 100 mesh sieve to obtain lotus seed starch-lecithin complexes.

complexes Compared with physical modification techniques using thermal processing, physical modification techniques without heat treatment, especially dynamic high pressure microfluidization (DHPM), are less well studied. DHPM, a new rapid and efficient dynamic pressure treatment technology, is widely used in manufacturing nano-microemulsions, nano-suspensions and liposomes (Li et al., 2018; Oliete, Potin, Cases, & Saurel, 2018). Our previous study has shown that the formation of starch-monoglyceride complexes by DHPM significantly affects the structural and physicochemical properties of the starch (Chen, Guo, Miao, Zeng, Jia et al., 2018; Chen, Guo, Zeng et al., 2018; Chen, Guo, Miao, Zeng, Guo et al., 2018). The formation of starch-lipid complexes is related to many factors, especially the processing methods and lipid types. Therefore, we hypothesized that a lotus seed starchlecithin complex with a stable structure could be obtained with proper DHPM pressure and lecithin concentration. Thus, our objective was to investigate the correlation between processing conditions and structural properties of lotus seed starch-lecithin complexes by dynamic high-pressure homogenization and to explore the formation mechanism of complexes under different homogenization pressure. The morphological characteristics were observed by field emission scanning electron microscopy (FESEM), and the molecular structure was assessed with size-exclusion chromatography combined with small-angle laser light scattering and refractive index (SEC-SALLS-RI). X-ray diffraction (XRD) was performed to investigate the crystal properties. The ordered structure was determined by Fourier transform infrared (FTIR) spectroscopy and 13C cross-polarization and magic-angle spinning nuclear magnetic resonance (13C CP/MAS NMR) spectroscopy. Furthermore, a visual correlation between processing conditions and structural properties was established in R software. The presented data provide new insight into the mechanism of lotus seed starch-lecithin complex formation.

2.3. Determination of the complex index According to the method of Chen, Guo, Miao, Zeng, Jia et al. (2018), Chen, Guo, Zeng et al. (2018), Chen, Guo, Miao, Zeng, Guo et al. (2018), 0.3 g of starch sample was weighed, dispersed in 5 ml of distilled water and boiled in a water bath for 20 min. After centrifugation (5000g, 10 min), 50 μl of the supernatant was extracted and mixed with 4 ml of dilute iodine solution (0.1% w/w) I2 and 2% (w/w) KI). The sample was analyzed with an ultraviolet spectrophotometer (UV-6100, Shanghai Meipuda Instrument Co., Ltd., Shanghai, China), and the absorbance of the sample at 610 nm was measured. The complex index (CI) was calculated with the formula below. The control sample was a starch sample without lecithin.

CI(%) = 100 ×

ABScontrol − ABSsample ABScontrol

where CI is the complex index of lotus seed starch and lecithin, ABScontrol is the absorbance of blank samples without lecithinin the same pressure group, and ABSsample is the absorbance of samples containing lecithin in the same pressure group. 2.4. FESEM measurements Samples were dispersed and fixed on a metal platform with a conductive double-sided adhesive, then subjected to gold spray treatment to attach a coating of approximately 50 nm to the sample. The sample was then transferred to a field emission scanning electron microscope (Nova NanoSEM 230, FEI Czech Republic S.R.O Ltd., Brno, Czech Republic) and observed with a 6 kV electron beam in low vacuum mode. A representative photo of the starch granules was taken at 5000× magnification.

2. Materials and methods 2.1. Materials

2.5. Molecular weight determination

Lotus seed was obtained from Green Field Fujian Food Co., Ltd. (Sanming, China), and the starch was isolated with the method reported in our previous study (Zhang, Zeng, Wang, Zeng, & Zheng, 2014). The raw lotus seed starch contained 9.23 ± 0.01% moisture, 0.36 ± 0.01% lipids, 0.28 ± 0.02% protein and 0.31 ± 0.01% ash. Lecithin with apurity > 99% was purchased from Shanghai Macklin Biochemical Co, Ltd. (Shanghai, China). All other chemical reagents used in this study were of analytical grade.

2.5.1. Preparation of samples The samples dissolved in DMSO solution (containing 50 mmol/l LiBr) at a concentration of 1 mg/ml were loaded into titration bottles and then heated in a water bath and stirred for 2 h with a magnetic stirrer (MYP11-2A, Shanghai Meiyingpu Instrument & Meter Manufacturing Co., Ltd., Shanghai, China) at a temperature of 95 °C. After heating, the sample was further stirred at room temperature for 24 h. The fully dissolved sample was centrifuged (5000 g, 10 min), and the supernatant was extracted. The sample was filtered through a nylon microporous membrane (0.45 μm) and subsequently analyzed with a SEC-SALLS-RI system (Viscotek TDA305max, Malvern Panalytical Ltd., Malvern, UK).

2.2. Preparation of lotus seed starch-lecithin complexes The raw starch was suspended in ethanol solution to completely remove lipids, and was then washed several times with distilled water. The defatted starch was dried in an air oven at 45 ± 1 °C for 16 h. 15 g of lotus seed starch was placed in a 250 ml beaker, and deionized water was added to prepare a 5% (w/w) starch emulsion. Then the solution was placed in a water bath at 50 °C, and 0%, 1% or 5% lecithin (based on the dry weight of lotus starch) was mixed with the starch emulsion during the stirring process (100 r/min, 10 min). After the aqueous suspension had completely dispersed, the sample solution was placed in a microfluidization apparatus (SPCH-10, Stansted Fluid Power Ltd., Harlow, UK) for high pressure homogenization; the initial temperature was 25 °C, the homogenization pressure was 60, 90 or 120 MPa, and the number of cycles was five. The DHPM was equipped with a circulating cooling water apparatus, which controlled the temperature of the machine cavity to 25 °C. Subsequently, the complex was centrifuged (5000g, 10 min), washed three times with 75% ethanol aqueous solution and then dried in a freeze dryer (FDU-1200, Tokyo Rikakikai Co., Ltd., Tokyo, Japan) for 18 h. The dried starch complexes were

2.5.2. Chromatographic analysis conditions The standard product was Pullulan produced by Shodex Ltd. (Tokyo, Japan). The size-exclusion system consisted of a chromatographic column (I-MBHMW-3078; Malvern Instruments Ltd., Malvern, England) and guard column (I Guard; Malvern Instruments Ltd., Malvern, England). The maximal molecular weight of the separation was 10 × 106 g/mol. The detector wavelength was 670 nm, and the detector temperature and column temperature were 50 °C. DMSO solution (containing 50 mmol/l LiBr) was used as the mobile phase, the flow rate was 0.5 ml/min, the injection volume was 100 μl, and dn/dc was 0.074. The experimental data were collected and calculated in OmniSec 5.0 to obtain the weight average molecular weight (Mw), number average molecular weight (Mn) and dispersion coefficient (Mw/Mn). 2

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1.094 ± 0.002c 1.098 ± 0.002f 15.68 ± 0.01c 2.84 ± 0.02c 1.108 ± 0.003b 1.064 ± 0.002 g 13.97 ± 0.01 g 2.16 ± 0.01 g 1.129 ± 0.002a 1.023 ± 0.002 h 12.31 ± 0.01 h 1.27 ± 0.01 h 1.068 ± 0.002d 1.104 ± 0.001e 13.94 ± 0.02 g 2.15 ± 0.01 g 1.031 ± 0.003f 1.145 ± 0.001b 15.36 ± 0.02d 2.75 ± 0.02d 1.035 ± 0.002f 1.131 ± 0.001c 15.25 ± 0.02e 2.64 ± 0.02e 1.043 ± 0.002 g 1.121 ± 0.002d 14.43 ± 0.01f 2.31 ± 0.01f

Microcrystalline\;region(% )= 100 ×

Ic1 Ic1 + Ic 2 + Ia

Subcrystalline\;region\;(% )= 100 ×

Ic 2 Ic1 + Ic 2 + Ia

Amorphous\;region\;(% )= 100 ×

Ia Ic1 + Ic 2 + Ia

where Ic1 is the area of the microcrystalline region, Ic2 is the area of the subcrystalline region, and Ia is the area of the amorphous region. 2.7. FTIR spectroscopy The sample was mixed with KBr powder in an agate mortar (1:100, w/w) and rapidly milled under an infrared lamp. Subsequently, the ground powder was placed in a vacuum mold and pressedinto a sheet, which was placed in a Fourier transform infrared spectroscope (Avatar360, Thermo Nicolet Corporation Ltd., Madison, US) for measurement. The scanning range was 4000–400 cm−1, the number of scans was 16–32, and the resolution was 4 cm−1. The automatic baseline correct was selected, and the smoothing point of the infrared spectrum is set to 5. The deconvolution process was performed on the infrared spectrum in the range of 950–1200 cm−1, and the deconvolution parameter was follows: 26 of half-peak width and an enhancement factor of 1.5. Fourier deconvolution, half-peak width and enhancement factor.

Different letters in the same row represent significant differences between different treatments (p < 0.05).

1.055 ± 0.002e 1.125 ± 0.002d 16.02 ± 0.01b 3.40 ± 0.01b

23.55 ± 0.01f 27.11 ± 0.02c 27.06 ± 0.02d 27.57 ± 0.01a

0.002d 0.001d 0.002a 0.001f 0.003c 0.02d ± ± ± ± ± ± 3.426 1.041 5.814 1.035 5.618 10.56

The sample was measured with an X-ray diffractometer (Xpert3, Analyx Corp. Ltd., Boston, US). The measurement parameters were as follows: tube pressure of 40 kV, current of 100 mA, scanning measurement in the range of 2θ = 0°–35° and scanning speed 8°/min. The division of microcrystalline and subcrystalline regions and the method of integration was referenced by the study (Chen et al., 2019), and the crystallinity of the starch sample was calculated by fitting the peak area in Peakfit 4.0. The formulas were as follows:

1.032 ± 0.002f 1.163 ± 0.002a 17.75 ± 0.01a 4.35 ± 0.01a

24.71 ± 0.11e g

21.11 ± 0.02 h

20.28 ± 0.02 24.95 ± 0.01e

3.280 ± 0.002f 1.054 ± 0.001c 5.114 ± 0.001c 0.825 ± 0.003 h 6.198 ± 0.003b 8.62 ± 0.02f

27.42 ± 0.02b

3.008 ± 0.002 g 1.073 ± 0.001a 3.578 ± 0.002f 0.548 ± 0.002i 6.529 ± 0.002a 5.75 ± 0.01i

26.9 ± 0.4b 3.648 ± 0.006a 3.552 ± 0.005a 1.027 ± 0.001f 2.975 ± 0.003 h 1.786 ± 0.002a 1.665 ± 0.002i 8.25 ± 0.01 g 15.1 ± 0.2e 3.482 ± 0.005f 3.291 ± 0.003f 1.058 ± 0.001b 3.394 ± 0.002 g 0.985 ± 0.002 g 3.446 ± 0.003 g 6.51 ± 0.01 h 3.227 ± 0.003

41.6 ± 0.6a 3.585 ± 0.003c 3.451 ± 0.002c 1.039 ± 0.002d 4.773 ± 0.002e 1.403 ± 0.003b 3.402 ± 0.003 h 12.43 ± 0.02a 25.3 ± 0.3c 3.506 ± 0.003e 3.335 ± 0.003e 1.051 ± 0.002c 4.926 ± 0.002d 1.193 ± 0.002e 4.129 ± 0.002f 9.31 ± 0.02e 3.458 ± 0.001

24.1 ± 0.2d 3.607 ± 0.002b 3.495 ± 0.002b 1.032 ± 0.001e 5.798 ± 0.003b 1.251 ± 0.002c 4.634 ± 0.002e 11.53 ± 0.02b 23.9 ± 0.3d 3.581 ± 0.002c 3.476 ± 0.003c 1.034 ± 0.001e 5.810 ± 0.002a 1.243 ± 0.002d 4.674 ± 0.003d 11.36 ± 0.01c

CI (%) Mw1 (106 g/ mol) Mn1 (106 g/mol) Mw1/Mn1 Mw2 (105 g/mol) Mn2 (105 g/mol) Mw2/Mn2 Microcrystalline region (%) Subcrystalline region (%) 995/1022 ratio 1047/1022 ratio C1 region (%) C4 region (%)

3.566 ± 0.006d

LS-5% Lecithin60 MPa LS-1% Lecithin60 MPa LS-0% Lecithin60 MPa

Table 1 Structural parameters of starch-lecithin complexes.

LS-0% Lecithin90 MPa

g

LS-1% Lecithin90 MPa

LS-5% Lecithin90 MPa

LS-0% Lecithin120 MPa

h

LS-5% Lecithin120 MPa LS-1% Lecithin120 MPa

2.6. XRD measurements

2.8. Solid-state

13

C CP/MAS NMR spectroscopy

The samples were measured as in the previous report (Zeng, Chen et al., 2018; Zeng, Zheng et al., 2018). Briefly, 0.3 g of complex powder was placed in a13C nuclear magnetic resonance spectrometer (AVANCE III 500, BrukerLtd., Karlsruhe, Germany) for scanning. The resonance frequency was 125.7 MHz, the MAS VTN 4 mm probe was used, the spectral width was 4 kHz, the spectral line was widened by 50 Hz, the acquisition time was 50 ms, the time domain was 2 k, and the conversion size was 4 k. Each spectrum was scanned 2400 times. The vibration intensity of the C1 region reflected the degree of crystallization of the starch crystal region, and the vibration intensity of the C4 region reflected the degree of amorphousness of the starch granules. The C1 and C4 peak areas of the starch samples were fitted and calculated in Peakfit 4.0. The formulas were as follows:

C1 region\;(%) = 100 ×

C1 Ctotal

C4 region\;(%) = 100 ×

C4 Ctotal

where C1 is the peak area of the vibration peak in the C1 region, C4 is the peak area of the vibration peak in the C4 region, and Ctotal is the total area of the vibration peaks. 2.9. Statistical analyses Graphs were constructed in OriginPro 9.0 (OriginLab Corporation, Northampton, MA, USA). Triplicate measurements were performed for 3

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starch granules with added lecithin weakened significantly, and the particle damage also decreased (green arrow in Fig. 1 E and F). This maybe because that the lotus seed starch was combined with lecithin at that time, and the formation of lotus seed starch-lecithin complexes might inhibit starch retrogradation by comparison with the apparent morphology of other retrograded starches. Moreover, when the homogenization pressure reached 120 MPa, the effects of lotus seed starchlecithin complex on inhibiting gelatinization became more significant as the concentration of lecithin increased. The morphology of the particles gradually recovered from sheets to ellipsoids (yellow arrow in Fig. 1H and I). These results demonstrated that the complexes protected the integrity of the particles and decreased starch gelation during the experiment, thus indicating that lotus seed starch-lecithin complexes played a key role in inhibiting the gelatinization and retrogradation of starch granules, in accordance with the previous results for high maize amylose-stearic acid complexes (Ocloo, Minnaar, & Emmambux, 2016). The high maize amylose-stearic acid complexes formed by gamma irradiation decrease the gelatinization of starch and resist breakage.

each experiment. The experimental data were analyzed for significance in DPS9.5 (Science Press, Beijing, China) (p < 0.05). A correlation matrix was visualized with Pearson correlation coefficient in R software version 3.5.1 and RStudio (R Package, USA). 3. Results and discussion 3.1. Complex index of starch-lecithin complexes The CI indirectly reflected the degree of complexation of amylose with fatty acids (Chen, Guo, Zeng et al., 2018). From Table 1, when the homogenization pressure was 60 MPa, the CI was 23.9 ± 0.3% and 24.1 ± 0.2% at lecithin concentrations of 1% and 5%. As the homogenization pressure increased to 90 MPa, the CI of samples increased to 25.3 ± 0.3% and 41.6 ± 0.6%, respectively, but when the pressure further increased to 120 MPa, the CI was 10.2 ± 0.1% and 14.7 ± 0.2% lower than those of the 90 MPa group. These data showed that different lecithin concentrations did not significantly affect starch complexes at low homogenization pressure, thus indicating that the low homogenization pressure was not sufficient to destroy the granular structure of lotus starch to enable elution of single-helix amylose, and a small amount of amylose was combined with lecithin. The results were similar to those reported by (Chen et al., 2017). Low pressure was not conducive to the formation of lotus seed starch-glycerin monostearin complexes. There were significant differences between the CI of samples obtained at 60 MPa and at 90 MPa and lecithin concentration of 1% from Table 1. It indicated that when the homogenization pressure increased to 90 MPa, the increasing starch granules were destroyed, thus the amylose had more opportunities to contact lecithin in the continuous phase, and the high-pressure shear force promoted the formation of complexes between lotus seed starch and lecithin. However, when the homogenization pressure was further raised to 120 MPa, the excessive homogenization pressure severely affected the hydrophobic cavity structure inside the amylose, or even destroyed the single helical structure of amylose, thus resulting in a decreased ability to form complexes with fatty acids, in agreement with the reported results (Chen et al., 2017).

3.3. Molecular structure of starch-lecithin complexes To explore the molecular structure, we used SEC-SALLS-RI to determine the molecular weight and polydispersity. As shown in the Fig. 2A, D and G, clear bimodal structures (Peak1 and Peak2) were observed in the differential signal map of lotus seed starch, thus indicating the presence of two different materials in the starch sample, which were considered to be amylopectin and amylose. The results were consistent with those of the previously reported study (Zeng et al., 2015). Two peaks in other figures indicated amylopectin, amylose and amylose-lecithin complexes; as verified by the previous paper (Chen, Guo, Miao, Zeng, Jia et al., 2018; Chen, Guo, Zeng et al., 2018; Chen, Guo, Miao, Zeng, Guo et al., 2018), in which the stearic acid was added resulted in a new peak by gel permeation chromatography, caused by the complex. Furthermore, as shown in Table 1, the molecular weight of amylopectin and amylose from lotus seed decreased with increasing pressure of microfluidization, in accordance with the reported results (Wei, Cai, Jin, & Tian, 2016). The molecular weight and polydispersity of waxy maize starch depended on the intensity of the homogenization pressure. Interestingly, the molecular weight of peak1 from lotus seed increased with the addition of lecithin at the same microfluidization pressure, whereas the molecular weight of peak2 declined with the addition of lecithin, possibly because the degradation of amylopectin was decreased by the formation of the complexes. The molecular weight of the complexes was lower than that of amylose with double helix structure, in which lecithin inhibited single helix amylose from forming a double helix structure. The results were also consistent with the morphological properties observed by FESEM. These results revealed that the formation of lotus seed starch-lecithin complexes contributed to the stability of the starch granule structure and significantly inhibited of amylopectin degradation and amylose retrogradation, in accordance with the findings of the previous paper (Li et al., 2014). The amylopectin with short chain or amylose formed by the degradation of amylopectin contributed to amylose retrogradation. The complex inhibited the amylose retrogradation partially by inhibiting the degradation of amylopectin. In this experiment, wheat starch-lipid complexes prevented formation of the non-covalent bond structure of the starch polymer under high pressure homogenization conditions. On the basis of the polydispersity, the Mw/Mn values of peak1 were close to 1, thus indicating a uniform composition. In contrast, the Mw/Mn values of peak2 were greater than 1, thus indicating that they contained some non-uniform components, such as single helix amylose, double helix amylose and amylose-lecithin complexes.

3.2. Morphological characteristics of starch-lecithin complexes The morphological images of lotus seed starch-lecithin complexes at 5000× magnification under different conditions are shown in Fig. 1. The natural lotus seed starch particles were mostly spherical or elliptical (Zhang et al., 2014). At 60 MPa, the surface of the lotus starch showed different degrees of deformation (red arrow in Fig. 1A), the particle shape remained intact, and the overall structure was stable. When the homogenization pressure was raised to 90 MPa or 120 MPa, the deformation of starch granules was clear, especially under 120 MPa pressure. A rough outer surface and granule shards of lotus seed starch were observed (red arrow in Fig. 1D and G). These results indicated that the extent to which the starch structure was destroyed increased as the homogenization pressure increased, in accordance with the findings for homogenization-treated potato and cassava starch (Che et al., 2009). When lecithin was added to the starch system, a small amount of an oily substance appeared on the surfaces of the starch granules under 60 MPa pressure (blue arrow in Fig. 1B and C), a result that became more pronounced as the concentration of lecithin increased. These results suggested that the lipids did not completely combine with the starch molecules, but part of the lecithin molecule was dissociated between the starch molecules. This phenomenon was consistent with the lotus seed starch-lipid complexes prepared under lower hydrostatic pressure (Jia, Sun, Chen, Zheng, & Guo, 2018). The lipids could not completely enter the starch granules under low force and were embedded in the gaps of the starch granules. When the pressure further increased to 90 MPa, samples displayed more oval starch granules compared to samples under 60 MPa, this was because that the retrogradation of lotus 4

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Fig. 1. Morphological images of lotus seed starch-lecithin complexes at 5000× magnification.

15.019°, 17.145°, 17.960°, 22.926°, and a new diffraction plane at 21.7° (red arrow), which was believed to be from the accumulation of free fatty acids between the starch chain spirals (Marinopoulou, Papastergiadis, & Raphaelides, 2016), thus indicating that the lotus seed-lecithin complexes displayed a V6II-type crystalline pattern. These results were consistent with the crystal structure of lotus seed starchglycerol monostearate (Chen et al., 2017). In contrast, the complexes with 1% and 5% lecithin formed at 90 MPa exhibited a different V6Itype crystalline pattern. The starch complexes changed from V6II type to V6I type crystal structure with increasing homogenization pressure, because the peak density of A-type diffraction decreased continuously with the increase of homogenization pressure, thus forming a stable Vtype crystalline pattern. Interestingly, the samples with 1% and 5% lecithin formed at 120 MPa displayed A- and V6II-type crystalline patterns, respectively. These results indicated that at high homogenization pressure, a large amount of lecithin was required to form V-type crystalline complexes. The complexes with 5% lecithin formed at 120 MPa showed diffraction planes at diffraction angles of 15.019°, 17.145°, 17.960°, 22.926°, thus indicating that the A-type crystallization region recovered partially. The proportions of the microcrystalline region and subcrystalline region of the complexes increased with increasing lecithin content (Table 1). When the homogenization pressure was 90 MPa and the lecithin content was 5%, the proportions of the microcrystalline region and crystallinity were the highest among all samples. These results demonstrated that complexes with stable structures formed under proper conditions, such as 90 MPa homogenization

3.4. Structural properties by XRD The crystal properties of lotus seed starch-lecithin complexes under different conditions determined by XRD, are shown in Fig. 3. Observed from Fig. 3A, D and G, after treatment with DHPM, lotus seed starch at the pressures of 60 , 90 and 120 MPa exhibited diffraction planes at diffraction angles of 15.019°, 17.145°, 17.960°, 22.926°, which was a typical crystalline pattern of A-type starch (Bettaïeb, Jerbi, & Ghorbel, 2014). But as the homogenization pressure increased, the proportions of the microcrystalline region and subcrystalline region of lotus seed starch decreased with increasing homogenization pressure (Table 1), especially in the microcrystalline region, as demonstrated by their molecular weights. These results indicated that the crystalline region of lotus seed starch may have been destroyed partially under the high pressure of DHPM, in agreement with the previously reported research (Guo et al., 2018; Meng, Ma, Cui, & Sun, 2014) that homogenization at high pressure would partially destroy the starch crystallization region, weakening the diffraction planes in the XRD pattern. Even when the processing intensity was further increased, the overall diffraction planes of starch would disappeared, leading to starch structure in an disorder. The effects of lecithin content on the XRD patterns of lotus seed starch-lecithin complexes are shown in Fig. 3 and Table 1. The starchlecithin complexes with 1% and 5% lecithin formed at 60 MPa showed diffraction planes at diffraction angles of 7.1°, 12.5°, 20.3° (blue arrow), a V-type crystalline pattern (Chen et al., 2017). Moreover, the complexes showed A-type crystal diffraction planes at diffraction angles of 5

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Fig. 2. Chromatograms of lotus seed starch-lecithin complexes by SEC-SALLS-RI system.

From Table 1, the 995/1022 ratio of lotus seed starch increased significantly with increasing homogenization pressure, thus indicating that formation of the double helix structure was promoted by homogenization pressure. This result was in line with the morphological properties observed by FESEM and the crystalline pattern observed by XRD. In contrast, with the addition of lecithin, the 995/1022 ratio of the complexes decreased, possibly because the formation of a single helix amylose complex with lecithin prevented the single helix structure from further forming a double helix structure. Moreover, the 1047/ 1022 ratio of samples at the same pressure increased with increasing lecithin content, a process related to the formation of starch-lecithin complexes with a single helix structure. These findings were consistent with the results for wheat starch-lysophosphatidylcholine complexes (Ahmadi-Abhari, Woortman, Hamer, Oudhuis, & Loos, 2013). The complexes formed under 90 MPa pressure with 5% lecithin displayed the highest 1047/1022 ratio among all samples, thus suggesting that the preparation conditions were beneficial for the formation of complexes with ordered structure. The ordered structure was consistent with the CI and crystallinity.

pressure and 5% lecithin, in agreement with the CI. These results were also consistent with the results for normal corn starch-lauric acid complexes (Chang et al., 2013), in which addition of lauric acid to the heated starch suspension resulted in the formation of more crystalline structure than that when lauric acid was added to the starch suspension and then heated.

3.5. Ordered structural properties by FTIR The ordered structural properties of lotus seed starch-lecithin complexes, determined by FTIR, are shown in Fig. 4(A). The infrared spectra of all samples had strong vibration peaks at 1047, 1081, 1022, 3320 and 2932 cm−1, which were generated by vibrations of the O–H, C–H, C–O, O–H and C–H bond valence of the molecular anhydroglucose unit, in agreement with the results of the previous study (Zheng, Zhang, & Zeng, 2016). When lecithin was added to the starch system, the lotus seed starch spectrum formed a new carbonyl vibration peak near the peak of 1738 cm−1 (blue arrow), which was considered to be the infrared characteristic stretching vibration peak of lecithin (Kuligowski, Quintás, Garrigues, & de la Guardia, 2008). It was suggested that the starch-lecithin complexes were obtained under the test pressure range. Furthermore, the bands at 995, 1022 and 1047 cm−1 were highly sensitive to changes in starch conformation. Among them, those at 995 and 1047 cm−1 reflected the stretching vibration of hydrated crystals and ordered structures, and that at 1022 cm−1 was the characteristic band of the amorphous regions (Zhang et al., 2014). Therefore, the ratio of relative peak intensity at the peaks of 1047 and 1022 cm−1 (1047/ 1022 ratio) was used to indicate the degree of order, and the ratio of relative peak intensity at the peaks of 995 and 1022 cm−1 (995/1022 ratio) was used to indicate the double helix degree (Chen et al., 2019).

3.6. Ordered structural characteristics determined by

13

C CP/MAS NMR

The ordered structural characteristics of lotus seed starch-lecithin complexes, determined by NMR, are shown in Fig. 4(B). After treatment with dynamic high-pressure homogenization, lotus seed starch at the pressures of 60, 90 and 120 MPa displayed the typical A-type crystalline pattern with two peaks at 101 and 100 ppm. With the addition of lecithin, the complexes displayed different spectra from lotus seed starch. The spectra of the complexes showed a weaker peak at 33.7 ppm (Fig. 4(B), blue arrow), which belonged to the fatty acid carbon chain of 6

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Fig. 3. XRD patterns of lotus seed starch-lecithin complexes.

properties by XRD and ordered structural properties by FTIR. Furthermore, with the addition of lecithin, the C1 and C4 region values of the complexes were enhanced at the same pressure. Interestingly, the complexes prepared at 90 MPa with 5% lecithin displayed the highest values, thus suggesting that they had the highest crystallinity and the lowest proportion of double helix structure among the samples. The results were also verified by XRD and FTIR, and were in line with the CI. These results demonstrated that V-type complexes with ordered structure formed under the proper conditions of homogenization pressure and lecithin content. Excessive homogenization pressure was not conducive to the formation of ordered structure V6I complexes, in accordance with results for debranched high amylose maize starch-lauric acid complexes (Liu, Chi, Huang, Li, & Chen, 2019). The complexes formed under atmospheric pressure displayed more V-type crystalline structure than those formed under high pressure.

lecithin (Zabar, Lesmes, Katz, Shimoni, & Bianco-Peled, 2009). Furthermore, the peak corresponding to alkyl carbon atoms at 33.7 ppm of the complexes shifted upfield by 1.17 ppm compared to free lecithin, which indicated that it did not come from un-complexed lecithin (Cheng et al., 2015). The complexes prepared at 60 MPa with 1% lecithin, 60 MPa with 5% lecithin, and 120 MPa with 5% lecithin exhibited peaks at 103, 81, 71 and 60 ppm, respectively, typical peaks of the V-type crystalline pattern (Fig. 4(B), black arrow). In the C1 region, these samples exhibited three peaks at 103,101 and 100 ppm, and the C1 region moved toward the high magnetic field region, relative to native starch. These were thought to have a V6II-type crystalline pattern. The results were consistent with those in the previous paper, in which amylose-lipid complexes formed under low pressure treatment had a V6II-type crystal structure (Le Bail et al., 2013). Nevertheless, the complexes prepared with 1% and 5% lecithin at 90 MPa exhibited a V6I-type crystalline pattern with shape peaks in the C1 region (Fig. 4(B), red arrow), possibly because pressure either too low or too high was not conducive to the formation of ordered structure V6I complexes. The fatty acid carbon chain of the V6II complex did not completely enter the spiral cavity of the amylose but was embedded in the helical gaps of the amylose, thus resulting in higher mobility of the fatty acid carbon chain. Moreover, the samples prepared with 1% lecithin at 120 MPa exhibited a A-type crystalline pattern, thus suggesting that V-type crystal complexes did not easily form under high homogenization pressure with a low lecithin content. Indeed, the C1 and C4 region values of lotus seed starch decreased with increasing homogenization pressure (Table 1), thus indicating that the crystallinity of the starch decreased and the proportion of double helix structure increased. The results were consistent with the crystal

3.7. Correlation analysis and formation mechanism of lotus seed starchlecithin complexes The Pearson correlation coefficient was used to visualize the relationship between the prepared conditions and structural properties of lotus seed starch-lecithin complexes, thus providing new insight into the formation mechanism of the complexes. In the heatmap in Fig. 5(A), positive correlations are displayed in red, and negative correlations in blue, and a lack of correlation is displayed in white. The color intensities of the squares are proportional to the correlation coefficients. Therefore, the homogenization pressure was positively correlated with 995/1022 ratio but negatively correlated with MW2, the 1047/1022 ratio, and microcrystalline and subcrystalline regions. Furthermore, the lecithin content was positively correlated with the 1047/1022 ratio, 7

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Fig. 4. Ordered structural properties of lotus seed starch-lecithin complexes by (A) FTIR and (B)

8

13

C CP/MAS NMR.

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Fig. 5. Visualized correlation and formation mechanism of lotus seed starch-lecithin complexes: (A) Heatmap of the correlation between processing conditions and structural properties; (B) The diagram of the formation mechanism of the complexes.

The formation mechanism of lotus seed starch-lecithin complexes by DHPM was then explored. The single-helical amylose from lotus seed was eluted from the starch granules on the basis of the high pressure shear and cavitation effects of microfluidization. After the addition of lecithin, the single-helical amylose and lecithin formed V-type complexes under the action of high pressure microfluidization. However,

and microcrystalline and subcrystalline regions, whereas it was negatively correlated with the 995/1022 ratio and MW2. There results were consistent with those of the structural analysis above. The Pearson correlation coefficient was effectively used to visualize the relationship between the preparation conditions and structural properties of lotus seed starch-lecithin complexes. 9

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draft. Zebin Guo: Investigation, Methodology. Baodong Zheng: Data curation, Funding acquisition, Supervision. Shaoxiao Zeng: Project administration, Resources, Software, Supervision. Hongliang Zeng: Project administration, Visualization, Writing - review & editing.

the lotus seed starch-lecithin complexes exhibited different structural characteristics under different preparation conditions. As shown in Fig. 5(B), at low homogenization pressure, the lower intensity homogenization pressure led to only a small fraction of amylose forming complexes with lecithin. Part of the lecithin was embedded in the gaps of the single helices of amylose, which was beneficial for the formation of low-density V6II complexes. In contrast, a stable V6I complex was formed at 90 MPa, because the higher pressure contributed to the dissolution of the single helix amylose, and the lecithin had more opportunities to enter the interior of the starch spiral cavity and combine with amylose to form stable V6II complexes. Interestingly, when the homogenization pressure was further increased to 120 MPa, the complexes exhibited V6II- or even A-type crystal structures. These results suggested that excessive homogenization pressure was not conducive to the formation of stable V6I complexes, possibly because excessive homogenization pressure further destroyed the single helix structure of amylose and prevented combination between starch and lecithin. These results revealed that stable lotus seed starch-lecithin complexes with V6I-type crystalline structure formed under appropriate processing conditions. The carbon chain in the lecithin molecule was mainly located in the hydrophobic cavity of the amylose, opposite to the methylene end; and the carboxyl group in the lecithin molecule was exposed outside the spiral cavity due to steric hindrance and electrostatic repulsion. The formation mechanism of lotus seed starch-lecithin was consistant with other lipid materials (Chen et al., 2017; Jia et al., 2018; Meng et al., 2014). The fatty acid carbon chain in the V6II complex did not completely enter the helical cavity of amylose, but was mostly embedded in the helical gap of amylose, while the fatty acid carbon chain in the V6I complex was completely encapsulated by the amylose spiral cavity.

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. Acknowledgements This work was supported by the Project of International Cooperation and Exchanges in Science and Technology of Fujian Agriculture and Forestry University (grant number KXGH17001), the National Natural Science Foundation of China (grant number 31871820 and 31701552), the Support Project for Distinguished Young Scholars of Fujian Agriculture and Forestry University (grant number xjq201714), the Program for Leading Talent in Fujian Provincial University (grant number 660160190), the Natural Science Foundation for Distinguished Young Scholars of Fujian Province (grant number 2019J06012) and Program for New Century Excellent Talents in Fujian Province University (grant number KLA18058A). References Ahmadi-Abhari, S., Woortman, A. J. J., Hamer, R. J., Oudhuis, A. A. C. M., & Loos, K. (2013). Influence of lysophosphatidylcholine on the gelation of diluted wheat starch suspensions. Carbohydrate Polymers, 93(1), 224–231. Bettaïeb, N. B., Jerbi, M. T., & Ghorbel, D. (2014). Gamma radiation influences pasting, thermal and structural properties of corn starch. Radition Physics and Chemistry, 103, 1–8. Chang, F. D., He, X. W., & Huang, Q. (2013). Effect of lauric acid on the V-amylose complex distribution and properties of swelled normal cornstarch granules. Journal of Cereal Science, 58, 89–95. Che, L. M., Wang, L. J., Li, D., Bhandari, B., Özkan, N., Chen, X. D., & Mao, Z. H. (2009). Starch pastes thinning during high-pressure homogenization. Carbohydrate Polymers, 75(1), 32–38. Chen, C. J., Fu, W. Q., Chang, Q., Zheng, B. D., Zhang, Y., & Zeng, H. L. (2019). Moisture distribution model describes the effect of water content on the structural properties of lotus seed resistant starch. Food Chemistry, 286, 449–458. Chen, B. Y., Guo, Z. B., Miao, S., Zeng, S. X., Jia, X. Z., Zhang, Y., & Zheng, B. D. (2018). Preparation and characterization of lotus seed starch-fatty acid complexes formed by microfluidization. Journal of Food Engineering, 237, 52–59. Chen, B. Y., Guo, Z. B., Zeng, S. X., Tian, Y. T., Miao, S., & Zheng, B. D. (2018). Paste structure and rheological properties of lotus seed starch glycerin monostearate complexes formed by high-pressure homogenization. Food Research International, 103, 380–389. Chen, B. Y., Jia, X. Z., Miao, S., Zeng, S. X., Guo, Z. B., Zhang, Y., & Zheng, B. D. (2018). Slowly digestible properties of lotus seed starch-glycerine monostearin complexes formed by high pressure homogenization. Food Chemistry, 252, 115–125. Chen, B. Y., Zeng, S. X., Zeng, H. L., Guo, Z. B., Zhang, Y., & Zheng, B. D. (2017). Properties of lotus seed starch–glycerin monostearin complexes formed by high pressure homogenization. Food Chemistry, 226, 119–127. Cheng, W. W., Luo, Z. G., Li, L., & Fu, X. (2015). Preparation and characterization of debranched-starch/phosphatidylcholine inclusion complexes. Journal of Agricultural And Food Chemistry, 63, 634–641. Guo, Z. B., Jia, X. Z., Miao, S., Chen, B. Y., Lu, X., & Zheng, B. D. (2018). Structural and thermal properties of amylose-fatty acid complexes prepared via high hydrostatic pressure. Food Chemistry, 264, 172–179. Haq, F., Yu, H. J., Wang, L., Teng, L. S., Haroon, M., Khan, R. U., ... Nazir, A. (2019). Advances in chemical modifications of starches and their applications. Carbohydrate Research, 476, 12–35. Jia, X. Z., Sun, S. W., Chen, B. Y., Zheng, B. D., & Guo, Z. B. (2018). Understanding the crystal structure of lotus seed amylose-long-chain fatty acid complexes prepared by high hydrostatic pressure. Food Research International, 111, 334–341. Kuligowski, J., Quintás, G., Garrigues, S., & de la Guardia, M. (2008). Determination of lecithin and soybean oil in dietary supplements using partial least squares–Fourier transform infrared spectroscopy. Talanta, 77(1), 229–234. Kumar, L., Brennan, M., Zheng, H. T., & Brennan, C. (2018). The effects of dairy ingredients on the pasting, textural, rheological, freeze-thaw properties and swelling behaviour of oat starch. Food Chemistry, 245, 518–524. Le Bail, P., Chauvet, B., Simonin, H., Rondeau-Mouro, C., Pontoire, B., de Carvalho, M., & Le-Bail, A. (2013). Formation and stability of amylose ligand complexes formed by high pressure treatment. Innovative Food Science & Emerging Technologies, 18, 1–6. Li, W. H., Cao, F., Fan, J., Ouyang, S. H., Luo, Q. G., Zheng, J. M., & Zhang, G. Q. (2014).

4. Conclusions The objective was to investigate the correlation between processing conditions and structural properties of lotus seed starch-lecithin complexes by dynamic high-pressure homogenization and to explore the formation mechanism. The lotus seed starch-lecithin complexes prepared at 90 MPa with 5% lecithin had the highest CI among these samples, and the complex protected the integrity of the particles and decreased starch retrogradation, as determined with FESEM. The molecular structure of the complex inhibited the degradation of amylopectin and retrogradation of amylose. The complex displayed different crystalline patterns (V6II type to V6I type then to V6II type) with increased homogenization pressure. The complex formed at 90 MPa with 5% lecithin had the highest the proportions of microcrystalline and subcrystalline regions among all samples. The results determined by FTIR and 13C CP/MAS NMR showed that the double helix degree was enhanced with increasing pressure, whereas the addition of lecithin contributed to the formation of an ordered structure of the single-helix amylose-lecithin complex, on the basis of the 995/1022 ratio and C1 region, because the formation of a single helix amylose complex with lecithin prevented the single helix structure to further form a double helix structure. Furthermore, correlation analysis indicated that the homogenization pressure was positively correlated with the 995/1022 ratio, whereas the lecithin content was positively correlated with the 1047/1022 ratio, microcrystalline and subcrystalline regions. The formation of stable lotus seed starch-lecithin complexes with V6I-type crystalline structure was accomplished under appropriate conditions. Too low or too high pressure was not conducive to the formation of ordered structures of single-helix amylose-lecithin complexes. These results should provide a theoretical basis for the development and utilization of starch-lipid complexes. CRediT authorship contribution statement Yixin Zheng: Conceptualization, Formal analysis, Writing - original 10

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