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An insight into the retrogradation behaviors and molecular structures of lotus seed starch-hydrocolloid blends
Food Chemistry 295 (2019) 548–555
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
An insight into the retrogradation behaviors and molecular structures of lotus seed starch-hydrocolloid blends
T
Mingjing Zhenga,b, Han Sua, Qingxiang Youa, Shaoxiao Zenga,b,c, Baodong Zhenga,b,c, ⁎ ⁎ Yi Zhanga,b,c, , Hongliang Zenga,b,c, a
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China Fujian Province 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 Hydrocolloids Autoclaving Retrogradation Structure
The retrogradation behaviors and molecular structures of lotus seed starch (LS) combined with different hydrocolloids, chitosan (CS), guar gum (GG) and xanthan (XN), were investigated. Following an autoclave treatment, the storage modulus (G') value of LS-CS increased more rapidly than LS alone, indicating an increase in starch retrogradation. This might result from intermolecular interactions, increased ordered structure, decreased weight-average molecular weight (Mw) and greater leached amylose content in LS-CS system. The LS-GG and particularly LS-XN blends showed the opposite trend. The much greater Mw of LS-XN was mainly attributed to the lower retrogradation rate. The enwrapping effect of GG or XN on LS, as observed by confocal laser scanning microscopy, might retard their retrogradation by limiting the granules’ swelling and the amylose leaching. Overall, the changes in the interaction force, ordered structure, Mw, leached amylose and microstructure were related to retrogradation behaviors of LS-hydrocolloid blends following an autoclave treatment.
1. Introduction Lotus (Nelumbo nucifera Gaertn.) is a large aquatic herb that has been cultivated in Asia (China, Australia, India and Japan) for thousands of year (Bhat & Sridhar, 2008). The seeds of lotus are the commonly edible part used as food or herbal medicine (Zhang et al., 2015). They can be made into various food products that are commercially available in Asian markets, including porridge, flours, starch, canned foods, juices and beverages (Zhang et al., 2015; Zhu, 2017a). Autoclave treatments are common sterilization processes for lotus seed products, especially canned foods, juices and beverages. However, autoclaving tends to induce undesirable effects on the stability, shelf-life and consumer acceptance of products. Because lotus seeds have a high amylose content of up to 40.20% (w/w, dry basis), they are more likely to undergo retrogradation following an autoclave treatment (Zhang et al., 2015). Generally, amylose molecules aggregate and re-associate more readily than amylopectin owing to their linear molecular morphology and short chain lengths, which result in retrogradation (crystallization) (Morris, 1990; Zavareze & Dias, 2011). An increase in the number of short amylose and amylopectin molecules as a result of the high temperature and pressure in the autoclaving process, increases the likelihood that molecules align and aggregate, retrograding gradually into ⁎
ordered structures upon cooling (Mutungi et al., 2011; Wang, Li, Copeland, Niu, & Wang, 2015). The starch retrogradation of a food is highly dependent on its composition, such as proteins and hydrocolloids. Hydrocolloids can act as crystallization inhibitors to prevent starch retrogradation, including guar gum (GG) (Fu & Bemiller, 2017; Zhang, Gu, Zhu, & Hong, 2018), xanthan (XN) (Zhang et al., 2018), sodium alginate (Li, Wang, Chen, Liu, & Li, 2017), corn fiber gum (Qiu et al., 2015) and chitosan (CS) (Raguzzoni, Delgadillo, & Silva, 2016). However, the effects are greatly influenced by the specific hydrocolloids used (including types and molecular sizes), the specific starches and the processing condition (Fu & Bemiller, 2017; Lee, Baek, Cha, Park, & Lim, 2002). For example, GG had no effect on the retrogradation of a tapioca starch gel according to Janya and Sanguansri (2008) or could even accelerate the short-term retrogradation of starch according to Funami et al. (2005), but it was more effective in retarding corn starch retrogradation than carrageenans and cellulose derivatives (Fu & Bemiller, 2017). Under autoclaving conditions, little information is available to clarify the effects of hydrocolloids of starch retrogradation or the related mechanisms. According to Song, Min, Li, and Zhou (2012), the addition of three food gums (konjac-glucomannan, carrageenan and gellan) to starch paste could exert either a positive or negative effect on
Corresponding authors at: College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China. E-mail addresses: [email protected] (Y. Zhang), [email protected] (H. Zeng).
https://doi.org/10.1016/j.foodchem.2019.05.166 Received 24 February 2019; Received in revised form 20 May 2019; Accepted 23 May 2019 Available online 24 May 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2.2.3. NMR determination The autoclaved LS-hydrocolloid blends were stored at 4 °C for 10 h. Prior to solid-state 13C cross-polarization and magic angle spinning (CP/MAS) NMR experiments, the samples were freeze-dried at −81 °C and 9 Pa for 48 h until the moisture contents of samples were less than 10%, then milled to pass through a 100-mesh sieve. The 13C CP/MAS experiments were performed using an AVIII 400 MHz WB spectrometer (Bruker Inc., Rheinstetten, Germany) using the method of Zhang et al. (2014) with some modifications. The samples were determined at a 13C frequency of 100.62 MHz and equipped with a double-resonance H/X CP-MAS 4-mm probe. Sample spectra were accumulated at a spinning rate of 6 kHz. The contact time was 1.8 ms with a recycle delay of 2 s. The number of scans was 1600 lines/mm for each spectrum with an acquisition time 0.021 s. Relative proportions of C4 peaks (%) were calculated using Eq. (2):
starch retrogradation under different autoclaving and cooling conditions, but the interactions between starch and gums during the autoclave treatments are not well understood. Thus, the objective of the present study was to utilize the molecular structures to explain the effect different hydrocolloids (CS, GG and XN) on the retrogradation behaviors of lotus seed starch (LS) following autoclave treatment. The determination included the dynamic rheological properties, interaction force, weight-average molecular weight (Mw) and leached amylose content, as well as by using solid-state 13C Nuclear magnetic resonance spectroscopy (NMR) and confocal laser scanning microscopy (CLSM) data. 2. Materials and methods 2.1. Materials
Relative proportions of C4 peaks (%) = A C4/(A C1 + A C4 + A C2,3,5 + A C6)
Lotus seeds were purchased from Green Acres (Fujian) Food Co. Ltd. (Sanming, China). GG (CAS-No.9000-30-0, purity ≥ 99.0%, galactomannan ≥ 85.0%) was obtained from Shanghai Lichen Biotechnology Co. Ltd. (Shanghai, China). XN [Cat No. G8800, viscosity (1% KCl) ≥ 1200 mpa.s] was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). CS (CAS-9012-76-4, carboxylation greater than 60%, viscosity 10–80 mpa.s) was provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (HPLC grade) and fluorescein 5-isothiocyanate (FITC) were from Sigma Chemical Co., Ltd. (St. Louis, MO, USA).
∗ 100%
where AC1, AC4, AC2,3,5, AC6 represent the fitting area of C1, C4, C2,3,5 and C6 peak using the integration method of MestreNova software, respectively. 2.2.4. Size-exclusion chromatography coupled with a multi-angle laser lightscattering and refractive index (SEC-MALLS-RI) system A SEC (OHpak SB-G, SB-806 M HQ columns and SB-804 HQ columns, Shodex, Tokyo, Japan) coupled with a MALLS (632.8 nm, DAWN DSP, Wyatt Technology, CA, USA) and a RI (RI-101, Shodex, Tokyo, Japan) system was used to determine the Mw of starch samples based on the method of Zhang et al. (2014) with minor modifications. The autoclaved LS-hydrocolloid blends were freeze-dried and milled to pass through a 100-mesh sieve. A 20-mg sample was dissolved in 10 mL 90% dimethyl sulfoxide containing 50 mmol/L LiBr, followed by heating at 90 °C for 2 h and stirring at 37 °C for 24 h on a stirrer-heater module. The supernatants were filtered through 0.45-μm nylon syringe filters, and then 1-mL samples were injected into the SEC-MALLS-RI system. The refractive index was set as 0.066 mL/g, as usually reported for starch dissolved in dimethyl sulfoxide based solvents (Guo et al., 2015; Zhong, Yokoyama, Wang, & Shoemaker, 2006).
2.2. Methods 2.2.1. Preparation of starch and LS-hydrocolloid blends following an autoclave treatment Lotus seeds were used to prepare the defatted starch (LS; moisture content 8.47 ± 0.52%, protein content 0.98 ± 0.11%) following the method of Zhang, Zeng, Wang, Zeng, and Zheng (2014). For the LShydrocolloid blends in each experiment, aqueous dispersions of 0.2%, 0.4% and 0.6% for each hydrocolloid (w/w) were prepared. Then, the defatted LS (6%, w/w) was added into the dispersions. The LS-CS, LSGG and LS-XN blends were obtained following autoclaving at 121 °C for 30 min using high-pressure sterilization equipment (SYQ-DSX-280B, Shenan Medical Devices, Shanghai, China).
2.2.5. Leached amylose The leached amylose contents of LS and LS-hydrocolloid blends were determined using the method described by Qiu et al. (2015) with minor modifications. After autoclaving, the samples were cooled to room temperature for 15 min, diluted 10 times with distilled water, and centrifuged at 10,528×g for 20 min. The supernatant was separated to determine the leached amylose content using an iodine colorimetric reaction. In addition, the supernatants of LS alone and each LS/0.40% hydrocolloid system were selected to observe the structures of leached amylose using an atomic force microscope (AFM; Agilent 5500, Agilent Technologies, CA, USA). In total, 1 mL supernatant was dissolved in 10 mL deionized water. Subsequently, 10 μL of the diluted mixture was spread onto a freshly cleaved mica sheet. After drying at room temperature, the samples were scanned in tapping mode in air using silicon cantilevers. Scan sizes between 4 μm × 4 μm were obtained.
2.2.2. Dynamic rheological properties The rheological measurements were performed according to the method of Li et al. (2017) with some modifications. The autoclaved LShydrocolloid blends were immediately transferred to a rheometer (Physica MCR 301, Anton Paar GmbH, Stuttgart, Germany) with a plate and plate geometry (diameter of 50 mm, and gap of 1 mm). Before rheological determinations, the samples were equilibrated for ∼ 10 min at a test temperature (4 °C). The storage modulus (G′) evolution was recorded as a function of time for 10 h at 4 °C to monitor the short-term retrogradation processes of the blends. The oscillation strain and oscillation frequency were set at 0.1% and 6.28 Hz, respectively. To determine the interaction forces between the LS and hydrocolloids, 6.0-g LS (6%, w/w, dry basis) samples were dispersed in solutions containing 0.40 g of each hydrocolloid. Then, NaCl or urea was added to the LS-hydrocolloid blends to achieve NaCl or urea final concentrations of 0.6 mol/L. The blends with and without NaCl or urea were autoclaved as described above, and then immediately cooled to 25 °C and equilibrated for ∼30 min. The frequency sweep was conducted from 0.1 to 100 rad/s, at a 0.1% strain. The changes of G′ (ΔG′) were calculated using Eq. (1):
′ (t ) − G0′ (t )/ G0′ (t ) ∗ 100% ΔG′ (%) = G0.6
(2)
2.2.6. CLSM observation Treatments with LS alone and each LS/0.40% hydrocolloid system were characterized to observe the starch swelling behavior following heating in hot water at 85 °C for 5 min and the structures of gelatinized granules following autoclaving at 121 °C for 30 min using a CLSM (Leica TCS SP8X DLS, Heidelberg, Germany). And 6% native LS and each 0.4% hydrocolloid aqueous dispersions were adopt as control. According to our previous study (Zheng, Su, et al., 2019), the heating in hot water at 85 °C for 5 min would not induce the complete
(1)
where G′0.6 (t) represents the G′ of the blends with NaCl or urea at the frequency of t rad/s; G′0 (t) represents the G′ of the blends without NaCl or urea at the frequency of t rad/s. 549
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gelatinization of LS, which is helpful when observing the swelling behavior of starch. The experiments were conducted using the methods described by Qiu et al. (2015) with minor modifications. LS-hydrocolloid blends (100 μL) were each stained with a 0.2% (w/v) FITC (20 μL) stock solution. Then, the stained sample was deposited onto a microscope slide and covered with a cover slip, storing at 4 °C for 10 h. FITC was excited by an argon 488-nm laser. Emission was within 500–525 nm. The objective used for all experiments provided a 20× magnification. 2.2.7. Statistical analysis All the experiments were conducted in triplicate. The results were expressed as mean ± standard deviations. Data were evaluated by a one-way analysis of variance using the DPS 9.50 system (Science Press, Beijing, China) with a least significant differences test. Statistical significance was set at p < 0.05. 3. Results and discussion 3.1. Dynamic rheological properties of LS-hydrocolloid blends following an autoclave treatment A dynamic time sweep of LS samples was conducted following an autoclave treatment. The G'(t)/G'(0) values of LS and LS-hydrocolloid blends with different hydrocolloid concentrations during the short-term retrogradation of 10 h at 4 °C are shown in Fig. 1, in which G'(0) and G'(t) represents the initial value of the storage modulus and the instantaneous value of the storage modulus, respectively. The G'(t)/G'(0) value is often used to monitor the gelation or retrogradation behavior of starch, which increased with the prolongation of the sweeping time for the retrogradation of starch, and indicates a more solid-like state during storage (Chen, Ren, Zhang, Tong, & Rashed, 2015; Li et al., 2017). The G'(t)/G'(0) values of LS blends with 0.20% CS and 0.40% CS were much greater than that of LS without a CS addition, while that of LS with 0.60% CS was gradually greater than LS alone when stored at 4 °C for 4 h. This contradicted the results of Raguzzoni et al. (2016), in which chitosan appeared to delay the retrogradation of starch at a very early stage, while slightly increasing the long-term retrogradation of starch. The differences are probably related to the changes in interactions between chitosan and starch molecules owing to the autoclave treatments. Conversely, the G'(t)/G'(0) values of all of the LS-GG and LS-XN blends were lower than that of LS alone, and decreased as the hydrocolloid concentrations increased. This result was in accordance with those of previous study in which the addition of hydrocolloids, such as GG or XN, could inhibit the increase in the G' of starch gel during aging (Kim & Yoo, 2006; Nagano, Tamaki, & Funami, 2008). In addition, the G'(t)/G'(0) values of LS-XN started to level off after 2 h of storage at 4 °C, while those of the other blends were still rapidly increasing. This indicated that the addition of XN could promote the formation of a stable starch gel structure faster than CS and GG, and this effectively decreased the starch retrogradation. Thus, the retrogradation of LS following autoclaving could be promoted by CS and inhibited by GG or XN, depending on the hydrocolloid type and concentration, and the retrogradation time.
Fig. 1. Changes in the G'(t)/G'(0) values of LS and LS-hydrocolloid blends following an autoclave treatment. (A) LS-CS blends; (B) LS-GG blends; (C) LS-XN blends. Note: G'(0) and G'(t) represents the initial value of the storage modulus and the instantaneous value of the storage modulus, respectively.
3.2. Interaction forces between LS and different hydrocolloids LS and the 6% LS-0.40% hydrocolloid blends were selected to observe the changes in G' resulting from the addition of destabilizing reagents (NaCl and urea). As shown in Fig. 2, both NaCl and urea addition caused the significant decreases in the G' value of LS (ΔG′ < 0, around −45% and −89%, respectively). It indicated that the hydrogen bonding and electrostatic interactions were both the main forces of LS gel. With the addition of hydrocolloids, no significant difference was found in the ΔG′ values of LS-CS and LS alone, while the ΔG′ values of LS-XN and LS-GG with NaCl were much less than that of those blends with urea. The results suggested that CS could not influence the
The NaCl (an electrostatic interactions breaking agent) and urea (a hydrogen bond breaking agent) were able to reduce the G' of starch during frequency sweep tests, due to destabilize the molecular forces contributing to the formation or maintenance of the three-dimensional network structure (Li et al., 2017; Shi, Li, Wang, & Adhikari, 2012). Based on the retrogradation behavior of LS-hydrocolloid blends above, LS with 0.40% CS showed the greatest change in the G'(t)/G'(0) of LS. Also, with the addition of hydrocolloids at the 0.40% level, LS-GG and LS-XN displayed significant decreases in the G'(t)/G'(0) of LS. Therefore, 550
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Fig. 2. Changes in the storage modulus (ΔG') values of LS and LS-hydrocolloid blends supplemented with NaCl or urea. Notes: −0.6 N-NaCl represents blends with 0.6 mol/L NaCl. −0.6 N-urea represents blends with 0.6 mol/L urea.
structures and retrogradation behaviors of LS blends following autoclaving when compared with LS-GG and LS-XN blends.
molecular interaction forces of LS gel, both hydrogen bonding and electrostatic interactions have great effects on the stability of LS-CS. While the hydrogen bonding, rather than electrostatic interactions, was the main force involved in the formation and stability of LS-XN and LSGG blends. With the addition of NaCl, the electrostatic interaction between the Na+ cations and the negatively charged hydroxyl groups on the starch could effectively reduce the starch-water interactions and particle-particle interactions, resulting in the weaker gel networks and lower G' of starch (Li, Fayet, & Homer, 2013; Shi et al., 2012). According to previous study, the GG-water interaction and coating effect of GG could limit the starch-water interaction (Zheng, You, et al., 2019). This seems to interfere the formation of electrostatic interaction in LS/GG system, causing the effect of NaCl on ΔG′ values of LS-GG blends was less than LS alone. As for LS-XN, maybe the negatively charged XN interact with Na+, which weaken the effect of NaCl on LSXN blends and the XN-starch interaction, in turn increasing the amylose-water interaction and the ΔG′ values of LS-XN were higher than 0. With the addition of urea, the ΔG′ values of LS-XN and LS-GG were much less than LS alone and LS-CS, suggesting the decreases in the hydrogen bonds of LS with XN and GG addition. However, the enhanced formation of hydrogen bonds was found in LS-XN and LS-GG blends based on the FT-IR in our previous studies (Zheng, Su, et al., 2019; Zheng, You, et al., 2019). This might be due to the type of those hydrogen bonds was not belong to the intermolecular hydrogen bonding. Since urea was regarded to break the intermolecular hydrogen bonding but had no effect on the intramolecular hydrogen bonding (Mcgrane, Mainwaring, Cornell, & Rix, 2004). Moreover, those interaction limited the amylose leaching and amylose-amylose interaction through intermolecular hydrogen bonding, thus resulting in a decrease of ΔG′ values. At a frequency near 100 rad/s, the G' values of LS blends supplemented with urea were not consistent with the above descriptions, which might result from the system’s instability at the end of testing. The different interaction forces might be an underlying factor that affects the properties of LS when blended with different hydrocolloids following an autoclave treatment. As reported by Cai, Hong, Gu, and Zhang (2011), the electrostatic repulsions between potato starch and XN could reduce the pasting peak viscosity and greatly increase the pasting temperatures, while the electrostatic attractions of the combinations showed the opposite effects, which affected the stability of the network structures. Thus, the electrostatic interactions in LS/CS systems and LS alone might be attributed to the different molecular
3.3. NMR spectra of retrograded LS-hydrocolloid blends The NMR spectra of starch samples stored for 10 h at 4 °C were used to evaluate the ordered structures in retrograded LS-hydrocolloid blends following an autoclave treatment (Fig. 3). And the chemical shifts and relative proportions of the C4 peaks of the samples are presented in Table 1. Three peaks (101.60, 100.35 and 99.6 ppm) were observed in the C1 signal region of ∼100 ppm for native LS without any hydrocolloids and autoclaving-cooling treatment. Based on the crystalline structures characterized by solid state 13C CP/MAS NMR (Veregin, Fyfe, Marchessault, & Taylor, 1986), this phenomenon indicated that native LS presented characteristics of both the A-type and B-type crystal structures. With the autoclaving-cooling treatment, LS showed the B-type crystal structure, with double peaks at 103.04 and 99.94 ppm. The results were well consistent with those of a previous study (Zhang et al., 2014). Compared with native LS, significant variations in chemical shifts to 102–104 ppm were found in all retrograded LS and LS-hydrocolloid blends, and these were assigned to the amylose and the simple helix as suggested by Morrison, Law, and Snape (1993). However, with the addition of hydrocolloids, no chemical shifts in the C1 signal region were found for LS, suggesting that the hydrocolloids had no effects on the crystalline pattern or helical forms (Veregin et al., 1986). Starch retrogradation has been studied by 13C CP/MAS NMR, which is related to the signal of the C4 peak (reflecting the amorphous phase of starch) (Ambigaipalan, Hoover, Donner, & Liu, 2013; Zhu, 2017b). The proportion of the amorphous phase could be measured by the proportion of C4 peak fitting area relative to the total area of the spectrum, which decreases as the ordered structure numbers (double helices) increases during starch retrogradation (Zeng et al., 2017; Zhang et al., 2014). As shown in Table 1, the amorphous phase of LS after an autoclaving-cooling treatment increased from 4.66% to 7.45%, suggesting the ordered structure numbers of LS were decreased with autoclaving-cooling treatment. This might result from the ordered crystalline area of starch granules being completely disrupted by high temperature and high pressure during the autoclaving process, while the further recrystallization during the short cooling storage could not regain a more ordered structure, based on the behavior of hydrated starch molecules in excess water (Matignon & Tecante, 2017). The 551
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essentially linear polysaccharide with a smaller molecular weight (∼105–6) (BeMiller, 2018). The weight-average molecular weight (Mw) of LS and LS-hydrocolloid blends are presented in Table 1. The results showed that the Mw of LS decreased from 1.283 × 107 ± 6.32% to 1.049 × 107 ± 1.72% g/mol after autoclaving. This might due to the leaching starch molecules (especially amylose) by the autoclave treatment, resulting in a lower average Mw of LS (Ovando-Martínez, Whitney, Reuhs, Doehlert, & Simsek, 2013; Zhang et al., 2014). With the addition of CS, the Mw of LS decreased to 3.251 × 106–7.798 × 105 g/mol (p < 0.05). This might indicating that the more starch molecules leached in LS/CS system, allowing them to orderly align and aggregate more easily, which is likely related to its increased retrogradation phenomenon. Additionally, the Mw of LS decreased as the CS concentration increased, indicating that, theoretically, the degree of retrogradation for 6% LS-0.60% CS should be the greatest among the blends. However, the degree of retrogradation for 6% LS-0.60% CS was lower than those of 6% LS-0.20% CS and 6% LS-0.40% CS based on the G'(t)/G'(0) values and NMR data. This inconsistency might be caused by the increased viscosity in the system when the CS concentrations were greater, based on the pasting viscosity behavior of starch/chitosan composites (Chang, Jian, Yu, & Ma, 2010). The increased viscosity can inhibit the interactions between starch molecules, thus retarding the recrystallization and retrogradation of starch (Shi & Bemiller, 2002). With the addition of GG and XN, the Mw values of LS increased significantly (p < 0.05), which might result from the formation of intermolecular associations between starch molecules and these hydrocolloids. According to previous reports, XN and GG may form intermolecular associations with leached amylose molecules during pasting, which relates to their abilities to prevent phase separation and/ or retrogradation (Fu & Bemiller, 2017; Shi & Bemiller, 2002). Furthermore, the Mw values of LS-XN blends were much greater than those of LS-GG, which might contribute to their lower retrogradation levels. 3.5. Leached amylose of LS-hydrocolloid blends following an autoclave treatment Lotus starch contains a high amount of amylose (∼40%) (Zhang et al., 2015). Generally, amylose will leach out of gelatinized starch dispersions during thermal treatments. The leached amylose aggregates and forms a three-dimensional network embedded with swollen starch granules, which plays an important role in the further gelation and retrogradation of starch (Mariotti, Caccialanza, Cappa, & Lucisano, 2018). The leaching amylose levels of LS and LS-hydrocolloid blends are shown in Table 1. Following autoclaving, the leaching amylose content of LS increased 24.68%–46.90% with CS addition, and decreased with GG or XN addition, especially with the addition of 0.60% XN (decrease of ∼20%). Moreover, an increase in the CS concentration contributed to a greater leaching amylose content (p < 0.05), suggesting that the leaching of amylose from LS could be promoted by the addition of CS. With the addition of GG, the leaching amylose content of LS decreased but no significant difference was found among the different GG concentrations (p > 0.05). In the LS/XN system, until the addition of 0.40% XN (and up), the leached amylose content of LS significantly decreased with the addition of increasing XN concentrations compared with LS (p < 0.05). The diminished molecular weight and greater leached amylose content in the LS/CS system resulted in a greater flexibility, which led to increased reaggregation and rearrangements, while the increased Mw and lower leached amylose content hindered the contact associations between starch molecules in the LS/GG and LS/XN systems, which affected the retrogradation of starch (Kim & Yoo, 2006; Mariotti et al., 2018). Compared with LS, the leached amylose content of LS-XN was significantly decreased until the addition of 0.40% XN (and up). At the 0.40% concentration, significant differences were found in the leached amylose contents of LS-CS, LS-XN and LS-GG. Therefore, each LS/0.40% hydrocolloid system was used to observe the microstructures of
Fig. 3. 13C CP/MAS NMR spectra of native LS, retrograded LS and LS-hydrocolloid blends following autoclaving.
proportion of the amorphous phase of LS decreased with the addition of CS, and increased with the addition of GG or XN. This suggested that the addition of CS facilitated the formation of an ordered structure and promoted starch retrogradation, while that addition of GG or XN resulted in the opposite trends. These findings were consistent with the results of the rheological property analysis. However, a linear relationship between the concentration of each hydrocolloid and its ordered structure was not found; therefore, further studies were needed. 3.4. Mw of LS-hydrocolloid blends following an autoclave treatment Most starch granules are is composed of a mixture of two molecules: amylose and amylopectin. Amylopectin molecules are highly branched with average molecular weights greater than 107, whereas amylose an 552
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Table 1 Chemical shifts, relative proportions of the C4 peaks, Mw values and leached amylose contents of retrograded LS and LS-hydrocolloid blends following an autoclave treatment.
Native LS
LS 6% LS-0.20% CS 6% LS-0.40% CS 6% LS-0.60% CS 6% LS-0.20% GG 6% LS-0.40% GG 6% LS-0.60% GG 6% LS-0.20% XN 6% LS-0.40% XN 6% LS-0.60% XN
C1
C4
C2, 3, 5
C6
Relative proportions of C4 peak (%)
Mw (g/mol)
Leached amylose content (%)
101.60 100.35 99.6 103.04 99.94 103.17 99.83 103.04 98.21 103.13 99.89 103.16 99.99 102.83 100.23 102.83 100.31 103.23 99.8 103.02 102.64 102.99 97.75
82.45
72.75
61.82
4.66
1.283 × 107 ± 6.32%g
–
82.56
72.76
61.91
7.45
1.049 × 107 ± 1.72%h
27.10 ± 0.17d
82.56
72.82
61.84
6.86
3.251 × 106 ± 1.44%i
33.79 ± 0.80c
82.70
72.77
61.91
6.94
8.928 × 105 ± 1.60%j
37.73 ± 0.33b
82.51
72.79
61.92
7.29
7.798 × 105 ± 1.54%k
39.81 ± 0.32a
82.54
72.78
61.92
8.40
1.137 × 107 ± 1.82%f
24.88 ± 0.80e
82.54
72.90
61.91
7.93
1.692 × 107 ± 1.96%e
25.82 ± 0.33e
82.27
72.85
61.82
7.93
1.786 × 107 ± 1.73%d
25.83 ± 0.32e
82.57
72.78
61.89
7.74
3.905 × 107 ± 8.27%a
26.96 ± 0.80d
82.44
72.92
61.93
8.54
3.697 × 107 ± 9.84%b
25.79 ± 0.33e
82.48
72.87
61.84
8.84
3.494 × 107 ± 2.30%c
21.47 ± 0.32f
Different superscript letters indicate statistical significances between averages at p < 0.05.
Fig. 4. AFM of leached amylose in LS-hydrocolloid blends following an autoclave treatment. (A) LS without a hydrocolloid; (B) LS-CS blends; (C) LS-GG blends; (B) LS-XN blends. Scan size is 4 × 4 μm.
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an oval shape of varying sizes (Fig. 5A). And CG and XN aqueous dispersions showed weaker fluorescence (white circles) than GG aqueous dispersions (Fig. 5B–D). When heated at 85 °C for 5 min, compared with LS alone (Fig. 5E), the swollen starch granules (red circles in Fig. 5) increased and were larger in size than those in the starch dispersion that contained CS (Fig. 5F). By contrast, when LS granules were heated in both GG (Fig. 5H) and XN (Fig. 5G) solutions, a non-uniform layer of gum existed around the surfaces of the granules, and the swelling and leaching of the starch components was inhibited. Zhang et al. (2018) have reported that the presence of both XN and GG resulted in significant decreases in the swelling extent of corn starch, while arabic gum had little or no effect on the swelling of starch granules during heating. The efficient thickening property and formation of the protecting or lubricating “barrier”, in part, explains why GG and XN restricted the swelling of starch granules effectively as discussed above in relation to leached amylose (Qiu et al., 2015; Zhang et al., 2018). Additionally, the swelling behaviors of LS-hydrocolloid blends following autoclaving were probably attributed to the changes in the leached amylose contents, which play important roles in starch retrogradation (Kim & Yoo, 2006; Mariotti et al., 2018). With the autoclave-cooling treatment, as can be seen in Fig. 5I–L, the starch granules lost shape, swelled and gelatinized to form a continuous and glutinous network. The LS-CS gel (Fig. 5J) appeared to be more compact than the LS gel (Fig. 4I), which might be the result of a reinforced interaction force, such as electrostatic interactions, between LS and CS. The compact structure probably induced the significant increase in the G' value and retrogradation rate of LS. The structures of matrices of mixed pastes supplemented independently with the addition of GG and XN (Fig. 5K and L) showed weak networks with obvious cavities (white arrows) and intermolecular cross-links or association
leaching amylose by AFM (Fig. 4). When compared with LS alone (Fig. 4A), the leached amylose content of the LS/CS system (Fig. 4B) was much smaller (white arrow) and an increasing tendency for particle aggregation (red arrow) occurred following autoclaving. As discussed above, the increased number of leached amylose and amylopectin molecules resulted in the increasing molecular alignment and aggregation in the LS/CS system (Mutungi et al., 2011; Wang et al., 2015). By contrast, the leached amylose contents of the LS/GG and LS/XN systems (Fig. 4C and D) decreased. These structural findings of the AFM correlated with the measurements of leached amylose. Therefore, it was hypothesized that the leaching and aggregation of amylose were affected by different hydrocolloids. The results were consistent with those of previous studies, in which decreases in starch polymer leaching occurred in starch supplemented with GG, XN, corn fiber gum and other hydrocolloids (Qiu et al., 2015; Zhang et al., 2018). The viscosity of the continuous phase may be increased by the presence of hydrocolloids, which prevent the diffusion of starch polymers from starch granules (Funami et al., 2005). Additionally, hydrocolloids, such as XN, are capable of enwrapping the starch granules and physically stabilizing the particle by acting as a protecting or lubricating “barrier”, thus limiting the amount of leached starch polymer (Heyman, Depypere, Meeren, & Dewettinck, 2013). These results were corroborated by the Mw values. 3.6. CLSM of LS-hydrocolloid blends The 6% LS in the absence and presence of each hydrocolloid at 0.40% was selected to observe the swelling process of starch granules during heating and the structure of gelatinized granules following autoclaving using CLSM. Images are displayed in Fig. 5. Native LS showed
Fig. 5. CLSM images of LS and 6% LS-0.40% hydrocolloid blends. (A) native LS; (B) (B) CS; (C) GG; (D) XN; (E) LS without a hydrocolloid heated to 85 °C for 5 min; (F) LS-CS blends heated to 85 °C for 5 min; (G) LS-GG blends heated to 85 °C for 5 min; (H) LS-XN blends heated to 85 °C for 5 min; (I) LS without hydrocolloid gelatinized at 4 °C for 10 h following an autoclave treatment; (J) LS-CS blends gelatinized at 4 °C for 10 h following an autoclave treatment; (K) LS-GG blends gelatinized at 4 °C for 10 h following an autoclave treatment; (L) LS-XN blends gelatinized at 4 °C for 10 h following an autoclave treatment. 554
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(red arrows). Such networks appeared to trap more water molecules in cavities and increase the water-holding capacity of the starch gel, which resulted in lower rates of G' and starch retrogradation.
Guo, Z., Zeng, S., Lu, X., Zhou, M., Zheng, M., & Zheng, B. (2015). Structural and physicochemical properties of lotus seed starch treated with ultra-high pressure. Food Chemistry, 186(1), 223–230. Heyman, B., Depypere, F., Meeren, P. V. D., & Dewettinck, K. (2013). Processing of waxy starch/xanthan gum mixtures within the gelatinization temperature range. Carbohydrate Polymers, 96(2), 560–567. Janya, M., & Sanguansri, C. (2008). Effects of hydrocolloids and freezing rates on freezethaw stability of tapioca starch gels. Food Hydrocolloids, 22(7), 1268–1272. Kim, C., & Yoo, B. (2006). Rheological properties of rice starch–xanthan gum mixtures. Journal of Food Engineering, 75(1), 120–128. Lee, M. H., Baek, M. H., Cha, D. S., Park, H. J., & Lim, S. T. (2002). Freeze–thaw stabilization of sweet potato starch gel by polysaccharide gums. Food Hydrocolloids, 16(4), 345–352. Li, D., Fayet, C., & Homer, S. (2013). Effect of NaCl on the thermal behaviour of wheat starch in excess and limited water. Carbohydrate Polymers, 94(1), 31–37. Li, Q., Wang, Y., Chen, H., Liu, S., & Li, M. (2017). Retardant effect of sodium alginate on the retrogradation properties of normal cornstarch and anti-retrogradation mechanism. Food Hydrocolloids, 69, 1–9. Mariotti, M., Caccialanza, G., Cappa, C., & Lucisano, M. (2018). Rheological behaviour of rice flour gels during formation: Influence of the amylose content and of the hydrothermal and mechanical history. Food Hydrocolloids, 84, 257–266. Matignon, A., & Tecante, A. (2017). Starch retrogradation: From starch components to cereal products. Food Hydrocolloids, 68, 43–52. Mcgrane, S. J., Mainwaring, D. E., Cornell, H. J., & Rix, C. J. (2004). The role of hydrogen bonding in amylose gelation. Starch – Stärke, 56(3–4), 122–131. Morris, V. J. (1990). Starch gelation and retrogradation. Trends in Food Science & Technology, 1(90), 2–6. Morrison, W. R., Law, R. V., & Snape, C. E. (1993). Evidence for inclusion complexes of lipids with V-amylose in maize, rice and oat starches. Journal of Cereal Science, 18(2), 107–109. Mutungi, C., Onyango, C., Doert, T., Paasch, S., Thiele, S., Machill, S., ... Rohm, H. (2011). Long- and short-range structural changes of recrystallised cassava starch subjected to in vitro digestion. Food Hydrocolloids, 25(3), 477–485. Nagano, T., Tamaki, E., & Funami, T. (2008). Influence of guar gum on granule morphologies and rheological properties of maize starch. Carbohydrate Polymers, 72(1), 95–101. Ovando-Martínez, M., Whitney, K., Reuhs, B. L., Doehlert, D. C., & Simsek, S. (2013). Effect of hydrothermal treatment on physicochemical and digestibility properties of oat starch. Food Research International, 52(1), 17–25. Qiu, S., Yadav, M. P., Chen, H., Liu, Y., Tatsumi, E., & Yin, L. (2015). Effects of corn fiber gum (CFG) on the pasting and thermal behaviors of maize starch. Carbohydrate Polymers, 115(115), 246–252. Raguzzoni, J. C., Delgadillo, I., & Silva, J. A. L. D. (2016). Influence of a cationic polysaccharide on starch functionality. Carbohydrate Polymers, 150, 369–377. Shi, A. M., Li, D., Wang, L. J., & Adhikari, B. (2012). The effect of NaCl on the rheological properties of suspension containing spray dried starch nanoparticles. Carbohydrate Polymers, 90(4), 1530–1537. Shi, X., & Bemiller, J. N. (2002). Effects of food gums on viscosities of starch suspensions during pasting. Carbohydrate Polymers, 50(1), 7–18. Song, R., Min, H., Li, B., & Zhou, B. (2012). The effect of three gums on the retrogradation of Indica rice starch. Nutrients, 4(6), 425–435. Veregin, R. P., Fyfe, C. A., Marchessault, R. H., & Taylor, M. G. (1986). Characterization of the crystalline A and B starch polymorphs and investigation of starch crystallization by high-resolution carbon-13 CP/MAS NMR. Macromolecules, 19(4), 873–880. Wang, S., Li, C., Copeland, L., Niu, Q., & Wang, S. (2015). Starch retrogradation: A comprehensive review. Comprehensive Reviews in Food Science & Food Safety, 14(5), 568–585. Zavareze, E. D. R., & Dias, A. R. G. (2011). Impact of heat-moisture treatment and annealing in starches: A review. Carbohydrate Polymers, 83(2), 317–328. Zeng, H., Huang, C., Chen, P., Zheng, B., Liu, J., & Zhang, Y. (2017). Comparative study on the structural and physicochemical properties of flour and starch from Dioscorea opposita Thunb. Chinese Journal of Structural Chemistry, 36(11), 1825–1836. Zhang, Y., Gu, Z., Zhu, L., & Hong, Y. (2018). Comparative study on the interaction between native corn starch and different hydrocolloids during gelatinization. International Journal of Biological Macromolecules, 116, 136–143. Zhang, Y., Lu, X., Zeng, S., Huang, X., Guo, Z., Zheng, Y., Tian, Y., & Zheng, B. (2015). Nutritional composition, physiological functions and processing of lotus (Nelumbo nucifera Gaertn.) seeds: A review. Phytochemistry Reviews, 14(3), 321–334. Zhang, Y., Zeng, H. L., Wang, Y., Zeng, S. X., & Zheng, B. D. (2014). Structural characteristics and crystalline properties of lotus seed resistant starch and its prebiotic effects. Food Chemistry, 155(19), 311–318. Zheng, M., Su, H., Luo, M., Shen, J., Zeng, S., Zheng, B., ... Zhang, Y. (2019). Effect of hydrocolloids on the retrogradation of lotus seed starch undergoing an autoclaving–cooling treatment. Journal of Food Science, 84, 466–474. Zheng, M., You, Q., Lin, Y., Lan, F., Luo, M., Zeng, H., ... Zhang, Y. (2019). Effect of guar gum on the physicochemical properties and in vitro digestibility of lotus seed starch. Food Chemistry, 272, 286–291. Zhong, F., Yokoyama, W., Wang, Q., & Shoemaker, C. F. (2006). Rice starch, amylopectin, and amylose: Molecular weight and solubility in dimethyl sulfoxide-based solvents. Journal of Agricultural and Food Chemistry, 54(6), 2320–2326. Zhu, F. (2017a). Structures, properties, and applications of lotus starches. Food Hydrocolloids, 63, 332–348. Zhu, F. (2017b). NMR spectroscopy of starch systems. Food Hydrocolloids, 63, 611–624.
4. Conclusions The molecular structures and retrogradation behaviors of LS blended with different hydrocolloids following autoclaving were investigated. The G'(t)/G'(0) values suggested that the retrogradation of LS following an autoclave treatment could be promoted by the addition of CS and retarded by the addition of GG or XN. Based on the changes in G' after the separate addition of NaCl and urea, the electrostatic interactions were the main forces affecting the LS and LS-CS gel, but they were not involved in the formation of LS-GG and LS-XN blends. This indicated that different interaction forces might be underlying factors that affect the properties of LS blended with different hydrocolloids. Compared with LS alone, the lower Mw and increased leached amylose content in the LS-CS blends resulted in a greater tendency to form higher ordered structures and the large aggregates of particles as observed by NMR and AFM, respectively. However, LS-GG and LS-XN showed the opposite trends, and the greater Mw of LS-XN was mainly attributed to its lower retrogradation rate. Furthermore, the enwrapping effects of GG and XN on LS, and the weakened structures for gelatinized LS blends as observed by CLSM, were also attributed to their decreased retrogradation behaviors. These data may have useful applications in producing high-amylose starch/hydrocolloid blends in foods, especially under autoclaving conditions. Declaration of Competing Interest None. Acknowledgements This study was financially supported by the Project of International Cooperation and Exchanges in Science and Technology of Fujian Agriculture and Forestry University (Grant number KXGH17001), the Scientific Research Foundation of Graduate School of Fujian Agriculture and Forestry University (Grant number YB2018009), the National Natural Science Funds (Grant number 31701552), the Leading Talents Support Program of Science and Technology Innovation in Fujian Province (Grant number KRC16002A), the Support Project for Distinguished Young Scholars of Fujian Agriculture and Forestry University (Grant number xjq201714), and the Science and Technology Innovation Project of Fujian Agriculture and Forestry University (Grant number CXZX2017414). References Ambigaipalan, P., Hoover, R., Donner, E., & Liu, Q. (2013). Retrogradation characteristics of pulse starches. Food Research International, 54(1), 203–212. BeMiller, J. N. (2018). Starches: Molecular and granular structures and properties. In J. N. BeMiller (Ed.). Carbohydrate chemistry for food scientists (pp. 159–189). (3rd ed.). Elsevier Inc. Bhat, R., & Sridhar, K. R. (2008). Nutritional quality evaluation of electron beam-irradiated lotus (Nelumbo nucifera) seeds. Food Chemistry, 107(1), 174–184. Cai, X., Hong, Y., Gu, Z., & Zhang, Y. (2011). The effect of electrostatic interactions on pasting properties of potato starch/xanthan gum combinations. Food Research International, 44(9), 3079–3086. Chang, P. R., Jian, R., Yu, J., & Ma, X. (2010). Fabrication and characterisation of chitosan nanoparticles/plasticised-starch composites. Food Chemistry, 120(3), 736–740. Chen, L., Ren, F., Zhang, Z., Tong, Q., & Rashed, M. M. A. (2015). Effect of pullulan on the short-term and long-term retrogradation of rice starch. Carbohydrate Polymers, 115, 415–421. Fu, Z., & Bemiller, J. N. (2017). Effect of hydrocolloids and salts on retrogradation of native and modified maize starch. Food Hydrocolloids, 69, 36–48. Funami, T., Kataoka, Y., Omoto, T., Goto, Y., Asai, I., & Nishinari, K. (2005). Effects of non-ionic polysaccharides on the gelatinization and retrogradation behavior of wheat starch. Food Hydrocolloids, 19(1), 1–13.
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An insight into the retrogradation behaviors and molecular structures of lotus seed starch-hydrocolloid blends
T
Mingjing Zhenga,b, Han Sua, Qingxiang Youa, Shaoxiao Zenga,b,c, Baodong Zhenga,b,c, ⁎ ⁎ Yi Zhanga,b,c, , Hongliang Zenga,b,c, a
College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China Fujian Province 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 Hydrocolloids Autoclaving Retrogradation Structure
The retrogradation behaviors and molecular structures of lotus seed starch (LS) combined with different hydrocolloids, chitosan (CS), guar gum (GG) and xanthan (XN), were investigated. Following an autoclave treatment, the storage modulus (G') value of LS-CS increased more rapidly than LS alone, indicating an increase in starch retrogradation. This might result from intermolecular interactions, increased ordered structure, decreased weight-average molecular weight (Mw) and greater leached amylose content in LS-CS system. The LS-GG and particularly LS-XN blends showed the opposite trend. The much greater Mw of LS-XN was mainly attributed to the lower retrogradation rate. The enwrapping effect of GG or XN on LS, as observed by confocal laser scanning microscopy, might retard their retrogradation by limiting the granules’ swelling and the amylose leaching. Overall, the changes in the interaction force, ordered structure, Mw, leached amylose and microstructure were related to retrogradation behaviors of LS-hydrocolloid blends following an autoclave treatment.
1. Introduction Lotus (Nelumbo nucifera Gaertn.) is a large aquatic herb that has been cultivated in Asia (China, Australia, India and Japan) for thousands of year (Bhat & Sridhar, 2008). The seeds of lotus are the commonly edible part used as food or herbal medicine (Zhang et al., 2015). They can be made into various food products that are commercially available in Asian markets, including porridge, flours, starch, canned foods, juices and beverages (Zhang et al., 2015; Zhu, 2017a). Autoclave treatments are common sterilization processes for lotus seed products, especially canned foods, juices and beverages. However, autoclaving tends to induce undesirable effects on the stability, shelf-life and consumer acceptance of products. Because lotus seeds have a high amylose content of up to 40.20% (w/w, dry basis), they are more likely to undergo retrogradation following an autoclave treatment (Zhang et al., 2015). Generally, amylose molecules aggregate and re-associate more readily than amylopectin owing to their linear molecular morphology and short chain lengths, which result in retrogradation (crystallization) (Morris, 1990; Zavareze & Dias, 2011). An increase in the number of short amylose and amylopectin molecules as a result of the high temperature and pressure in the autoclaving process, increases the likelihood that molecules align and aggregate, retrograding gradually into ⁎
ordered structures upon cooling (Mutungi et al., 2011; Wang, Li, Copeland, Niu, & Wang, 2015). The starch retrogradation of a food is highly dependent on its composition, such as proteins and hydrocolloids. Hydrocolloids can act as crystallization inhibitors to prevent starch retrogradation, including guar gum (GG) (Fu & Bemiller, 2017; Zhang, Gu, Zhu, & Hong, 2018), xanthan (XN) (Zhang et al., 2018), sodium alginate (Li, Wang, Chen, Liu, & Li, 2017), corn fiber gum (Qiu et al., 2015) and chitosan (CS) (Raguzzoni, Delgadillo, & Silva, 2016). However, the effects are greatly influenced by the specific hydrocolloids used (including types and molecular sizes), the specific starches and the processing condition (Fu & Bemiller, 2017; Lee, Baek, Cha, Park, & Lim, 2002). For example, GG had no effect on the retrogradation of a tapioca starch gel according to Janya and Sanguansri (2008) or could even accelerate the short-term retrogradation of starch according to Funami et al. (2005), but it was more effective in retarding corn starch retrogradation than carrageenans and cellulose derivatives (Fu & Bemiller, 2017). Under autoclaving conditions, little information is available to clarify the effects of hydrocolloids of starch retrogradation or the related mechanisms. According to Song, Min, Li, and Zhou (2012), the addition of three food gums (konjac-glucomannan, carrageenan and gellan) to starch paste could exert either a positive or negative effect on
Corresponding authors at: College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China. E-mail addresses: [email protected] (Y. Zhang), [email protected] (H. Zeng).
https://doi.org/10.1016/j.foodchem.2019.05.166 Received 24 February 2019; Received in revised form 20 May 2019; Accepted 23 May 2019 Available online 24 May 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
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2.2.3. NMR determination The autoclaved LS-hydrocolloid blends were stored at 4 °C for 10 h. Prior to solid-state 13C cross-polarization and magic angle spinning (CP/MAS) NMR experiments, the samples were freeze-dried at −81 °C and 9 Pa for 48 h until the moisture contents of samples were less than 10%, then milled to pass through a 100-mesh sieve. The 13C CP/MAS experiments were performed using an AVIII 400 MHz WB spectrometer (Bruker Inc., Rheinstetten, Germany) using the method of Zhang et al. (2014) with some modifications. The samples were determined at a 13C frequency of 100.62 MHz and equipped with a double-resonance H/X CP-MAS 4-mm probe. Sample spectra were accumulated at a spinning rate of 6 kHz. The contact time was 1.8 ms with a recycle delay of 2 s. The number of scans was 1600 lines/mm for each spectrum with an acquisition time 0.021 s. Relative proportions of C4 peaks (%) were calculated using Eq. (2):
starch retrogradation under different autoclaving and cooling conditions, but the interactions between starch and gums during the autoclave treatments are not well understood. Thus, the objective of the present study was to utilize the molecular structures to explain the effect different hydrocolloids (CS, GG and XN) on the retrogradation behaviors of lotus seed starch (LS) following autoclave treatment. The determination included the dynamic rheological properties, interaction force, weight-average molecular weight (Mw) and leached amylose content, as well as by using solid-state 13C Nuclear magnetic resonance spectroscopy (NMR) and confocal laser scanning microscopy (CLSM) data. 2. Materials and methods 2.1. Materials
Relative proportions of C4 peaks (%) = A C4/(A C1 + A C4 + A C2,3,5 + A C6)
Lotus seeds were purchased from Green Acres (Fujian) Food Co. Ltd. (Sanming, China). GG (CAS-No.9000-30-0, purity ≥ 99.0%, galactomannan ≥ 85.0%) was obtained from Shanghai Lichen Biotechnology Co. Ltd. (Shanghai, China). XN [Cat No. G8800, viscosity (1% KCl) ≥ 1200 mpa.s] was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). CS (CAS-9012-76-4, carboxylation greater than 60%, viscosity 10–80 mpa.s) was provided by Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (HPLC grade) and fluorescein 5-isothiocyanate (FITC) were from Sigma Chemical Co., Ltd. (St. Louis, MO, USA).
∗ 100%
where AC1, AC4, AC2,3,5, AC6 represent the fitting area of C1, C4, C2,3,5 and C6 peak using the integration method of MestreNova software, respectively. 2.2.4. Size-exclusion chromatography coupled with a multi-angle laser lightscattering and refractive index (SEC-MALLS-RI) system A SEC (OHpak SB-G, SB-806 M HQ columns and SB-804 HQ columns, Shodex, Tokyo, Japan) coupled with a MALLS (632.8 nm, DAWN DSP, Wyatt Technology, CA, USA) and a RI (RI-101, Shodex, Tokyo, Japan) system was used to determine the Mw of starch samples based on the method of Zhang et al. (2014) with minor modifications. The autoclaved LS-hydrocolloid blends were freeze-dried and milled to pass through a 100-mesh sieve. A 20-mg sample was dissolved in 10 mL 90% dimethyl sulfoxide containing 50 mmol/L LiBr, followed by heating at 90 °C for 2 h and stirring at 37 °C for 24 h on a stirrer-heater module. The supernatants were filtered through 0.45-μm nylon syringe filters, and then 1-mL samples were injected into the SEC-MALLS-RI system. The refractive index was set as 0.066 mL/g, as usually reported for starch dissolved in dimethyl sulfoxide based solvents (Guo et al., 2015; Zhong, Yokoyama, Wang, & Shoemaker, 2006).
2.2. Methods 2.2.1. Preparation of starch and LS-hydrocolloid blends following an autoclave treatment Lotus seeds were used to prepare the defatted starch (LS; moisture content 8.47 ± 0.52%, protein content 0.98 ± 0.11%) following the method of Zhang, Zeng, Wang, Zeng, and Zheng (2014). For the LShydrocolloid blends in each experiment, aqueous dispersions of 0.2%, 0.4% and 0.6% for each hydrocolloid (w/w) were prepared. Then, the defatted LS (6%, w/w) was added into the dispersions. The LS-CS, LSGG and LS-XN blends were obtained following autoclaving at 121 °C for 30 min using high-pressure sterilization equipment (SYQ-DSX-280B, Shenan Medical Devices, Shanghai, China).
2.2.5. Leached amylose The leached amylose contents of LS and LS-hydrocolloid blends were determined using the method described by Qiu et al. (2015) with minor modifications. After autoclaving, the samples were cooled to room temperature for 15 min, diluted 10 times with distilled water, and centrifuged at 10,528×g for 20 min. The supernatant was separated to determine the leached amylose content using an iodine colorimetric reaction. In addition, the supernatants of LS alone and each LS/0.40% hydrocolloid system were selected to observe the structures of leached amylose using an atomic force microscope (AFM; Agilent 5500, Agilent Technologies, CA, USA). In total, 1 mL supernatant was dissolved in 10 mL deionized water. Subsequently, 10 μL of the diluted mixture was spread onto a freshly cleaved mica sheet. After drying at room temperature, the samples were scanned in tapping mode in air using silicon cantilevers. Scan sizes between 4 μm × 4 μm were obtained.
2.2.2. Dynamic rheological properties The rheological measurements were performed according to the method of Li et al. (2017) with some modifications. The autoclaved LShydrocolloid blends were immediately transferred to a rheometer (Physica MCR 301, Anton Paar GmbH, Stuttgart, Germany) with a plate and plate geometry (diameter of 50 mm, and gap of 1 mm). Before rheological determinations, the samples were equilibrated for ∼ 10 min at a test temperature (4 °C). The storage modulus (G′) evolution was recorded as a function of time for 10 h at 4 °C to monitor the short-term retrogradation processes of the blends. The oscillation strain and oscillation frequency were set at 0.1% and 6.28 Hz, respectively. To determine the interaction forces between the LS and hydrocolloids, 6.0-g LS (6%, w/w, dry basis) samples were dispersed in solutions containing 0.40 g of each hydrocolloid. Then, NaCl or urea was added to the LS-hydrocolloid blends to achieve NaCl or urea final concentrations of 0.6 mol/L. The blends with and without NaCl or urea were autoclaved as described above, and then immediately cooled to 25 °C and equilibrated for ∼30 min. The frequency sweep was conducted from 0.1 to 100 rad/s, at a 0.1% strain. The changes of G′ (ΔG′) were calculated using Eq. (1):
′ (t ) − G0′ (t )/ G0′ (t ) ∗ 100% ΔG′ (%) = G0.6
(2)
2.2.6. CLSM observation Treatments with LS alone and each LS/0.40% hydrocolloid system were characterized to observe the starch swelling behavior following heating in hot water at 85 °C for 5 min and the structures of gelatinized granules following autoclaving at 121 °C for 30 min using a CLSM (Leica TCS SP8X DLS, Heidelberg, Germany). And 6% native LS and each 0.4% hydrocolloid aqueous dispersions were adopt as control. According to our previous study (Zheng, Su, et al., 2019), the heating in hot water at 85 °C for 5 min would not induce the complete
(1)
where G′0.6 (t) represents the G′ of the blends with NaCl or urea at the frequency of t rad/s; G′0 (t) represents the G′ of the blends without NaCl or urea at the frequency of t rad/s. 549
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gelatinization of LS, which is helpful when observing the swelling behavior of starch. The experiments were conducted using the methods described by Qiu et al. (2015) with minor modifications. LS-hydrocolloid blends (100 μL) were each stained with a 0.2% (w/v) FITC (20 μL) stock solution. Then, the stained sample was deposited onto a microscope slide and covered with a cover slip, storing at 4 °C for 10 h. FITC was excited by an argon 488-nm laser. Emission was within 500–525 nm. The objective used for all experiments provided a 20× magnification. 2.2.7. Statistical analysis All the experiments were conducted in triplicate. The results were expressed as mean ± standard deviations. Data were evaluated by a one-way analysis of variance using the DPS 9.50 system (Science Press, Beijing, China) with a least significant differences test. Statistical significance was set at p < 0.05. 3. Results and discussion 3.1. Dynamic rheological properties of LS-hydrocolloid blends following an autoclave treatment A dynamic time sweep of LS samples was conducted following an autoclave treatment. The G'(t)/G'(0) values of LS and LS-hydrocolloid blends with different hydrocolloid concentrations during the short-term retrogradation of 10 h at 4 °C are shown in Fig. 1, in which G'(0) and G'(t) represents the initial value of the storage modulus and the instantaneous value of the storage modulus, respectively. The G'(t)/G'(0) value is often used to monitor the gelation or retrogradation behavior of starch, which increased with the prolongation of the sweeping time for the retrogradation of starch, and indicates a more solid-like state during storage (Chen, Ren, Zhang, Tong, & Rashed, 2015; Li et al., 2017). The G'(t)/G'(0) values of LS blends with 0.20% CS and 0.40% CS were much greater than that of LS without a CS addition, while that of LS with 0.60% CS was gradually greater than LS alone when stored at 4 °C for 4 h. This contradicted the results of Raguzzoni et al. (2016), in which chitosan appeared to delay the retrogradation of starch at a very early stage, while slightly increasing the long-term retrogradation of starch. The differences are probably related to the changes in interactions between chitosan and starch molecules owing to the autoclave treatments. Conversely, the G'(t)/G'(0) values of all of the LS-GG and LS-XN blends were lower than that of LS alone, and decreased as the hydrocolloid concentrations increased. This result was in accordance with those of previous study in which the addition of hydrocolloids, such as GG or XN, could inhibit the increase in the G' of starch gel during aging (Kim & Yoo, 2006; Nagano, Tamaki, & Funami, 2008). In addition, the G'(t)/G'(0) values of LS-XN started to level off after 2 h of storage at 4 °C, while those of the other blends were still rapidly increasing. This indicated that the addition of XN could promote the formation of a stable starch gel structure faster than CS and GG, and this effectively decreased the starch retrogradation. Thus, the retrogradation of LS following autoclaving could be promoted by CS and inhibited by GG or XN, depending on the hydrocolloid type and concentration, and the retrogradation time.
Fig. 1. Changes in the G'(t)/G'(0) values of LS and LS-hydrocolloid blends following an autoclave treatment. (A) LS-CS blends; (B) LS-GG blends; (C) LS-XN blends. Note: G'(0) and G'(t) represents the initial value of the storage modulus and the instantaneous value of the storage modulus, respectively.
3.2. Interaction forces between LS and different hydrocolloids LS and the 6% LS-0.40% hydrocolloid blends were selected to observe the changes in G' resulting from the addition of destabilizing reagents (NaCl and urea). As shown in Fig. 2, both NaCl and urea addition caused the significant decreases in the G' value of LS (ΔG′ < 0, around −45% and −89%, respectively). It indicated that the hydrogen bonding and electrostatic interactions were both the main forces of LS gel. With the addition of hydrocolloids, no significant difference was found in the ΔG′ values of LS-CS and LS alone, while the ΔG′ values of LS-XN and LS-GG with NaCl were much less than that of those blends with urea. The results suggested that CS could not influence the
The NaCl (an electrostatic interactions breaking agent) and urea (a hydrogen bond breaking agent) were able to reduce the G' of starch during frequency sweep tests, due to destabilize the molecular forces contributing to the formation or maintenance of the three-dimensional network structure (Li et al., 2017; Shi, Li, Wang, & Adhikari, 2012). Based on the retrogradation behavior of LS-hydrocolloid blends above, LS with 0.40% CS showed the greatest change in the G'(t)/G'(0) of LS. Also, with the addition of hydrocolloids at the 0.40% level, LS-GG and LS-XN displayed significant decreases in the G'(t)/G'(0) of LS. Therefore, 550
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Fig. 2. Changes in the storage modulus (ΔG') values of LS and LS-hydrocolloid blends supplemented with NaCl or urea. Notes: −0.6 N-NaCl represents blends with 0.6 mol/L NaCl. −0.6 N-urea represents blends with 0.6 mol/L urea.
structures and retrogradation behaviors of LS blends following autoclaving when compared with LS-GG and LS-XN blends.
molecular interaction forces of LS gel, both hydrogen bonding and electrostatic interactions have great effects on the stability of LS-CS. While the hydrogen bonding, rather than electrostatic interactions, was the main force involved in the formation and stability of LS-XN and LSGG blends. With the addition of NaCl, the electrostatic interaction between the Na+ cations and the negatively charged hydroxyl groups on the starch could effectively reduce the starch-water interactions and particle-particle interactions, resulting in the weaker gel networks and lower G' of starch (Li, Fayet, & Homer, 2013; Shi et al., 2012). According to previous study, the GG-water interaction and coating effect of GG could limit the starch-water interaction (Zheng, You, et al., 2019). This seems to interfere the formation of electrostatic interaction in LS/GG system, causing the effect of NaCl on ΔG′ values of LS-GG blends was less than LS alone. As for LS-XN, maybe the negatively charged XN interact with Na+, which weaken the effect of NaCl on LSXN blends and the XN-starch interaction, in turn increasing the amylose-water interaction and the ΔG′ values of LS-XN were higher than 0. With the addition of urea, the ΔG′ values of LS-XN and LS-GG were much less than LS alone and LS-CS, suggesting the decreases in the hydrogen bonds of LS with XN and GG addition. However, the enhanced formation of hydrogen bonds was found in LS-XN and LS-GG blends based on the FT-IR in our previous studies (Zheng, Su, et al., 2019; Zheng, You, et al., 2019). This might be due to the type of those hydrogen bonds was not belong to the intermolecular hydrogen bonding. Since urea was regarded to break the intermolecular hydrogen bonding but had no effect on the intramolecular hydrogen bonding (Mcgrane, Mainwaring, Cornell, & Rix, 2004). Moreover, those interaction limited the amylose leaching and amylose-amylose interaction through intermolecular hydrogen bonding, thus resulting in a decrease of ΔG′ values. At a frequency near 100 rad/s, the G' values of LS blends supplemented with urea were not consistent with the above descriptions, which might result from the system’s instability at the end of testing. The different interaction forces might be an underlying factor that affects the properties of LS when blended with different hydrocolloids following an autoclave treatment. As reported by Cai, Hong, Gu, and Zhang (2011), the electrostatic repulsions between potato starch and XN could reduce the pasting peak viscosity and greatly increase the pasting temperatures, while the electrostatic attractions of the combinations showed the opposite effects, which affected the stability of the network structures. Thus, the electrostatic interactions in LS/CS systems and LS alone might be attributed to the different molecular
3.3. NMR spectra of retrograded LS-hydrocolloid blends The NMR spectra of starch samples stored for 10 h at 4 °C were used to evaluate the ordered structures in retrograded LS-hydrocolloid blends following an autoclave treatment (Fig. 3). And the chemical shifts and relative proportions of the C4 peaks of the samples are presented in Table 1. Three peaks (101.60, 100.35 and 99.6 ppm) were observed in the C1 signal region of ∼100 ppm for native LS without any hydrocolloids and autoclaving-cooling treatment. Based on the crystalline structures characterized by solid state 13C CP/MAS NMR (Veregin, Fyfe, Marchessault, & Taylor, 1986), this phenomenon indicated that native LS presented characteristics of both the A-type and B-type crystal structures. With the autoclaving-cooling treatment, LS showed the B-type crystal structure, with double peaks at 103.04 and 99.94 ppm. The results were well consistent with those of a previous study (Zhang et al., 2014). Compared with native LS, significant variations in chemical shifts to 102–104 ppm were found in all retrograded LS and LS-hydrocolloid blends, and these were assigned to the amylose and the simple helix as suggested by Morrison, Law, and Snape (1993). However, with the addition of hydrocolloids, no chemical shifts in the C1 signal region were found for LS, suggesting that the hydrocolloids had no effects on the crystalline pattern or helical forms (Veregin et al., 1986). Starch retrogradation has been studied by 13C CP/MAS NMR, which is related to the signal of the C4 peak (reflecting the amorphous phase of starch) (Ambigaipalan, Hoover, Donner, & Liu, 2013; Zhu, 2017b). The proportion of the amorphous phase could be measured by the proportion of C4 peak fitting area relative to the total area of the spectrum, which decreases as the ordered structure numbers (double helices) increases during starch retrogradation (Zeng et al., 2017; Zhang et al., 2014). As shown in Table 1, the amorphous phase of LS after an autoclaving-cooling treatment increased from 4.66% to 7.45%, suggesting the ordered structure numbers of LS were decreased with autoclaving-cooling treatment. This might result from the ordered crystalline area of starch granules being completely disrupted by high temperature and high pressure during the autoclaving process, while the further recrystallization during the short cooling storage could not regain a more ordered structure, based on the behavior of hydrated starch molecules in excess water (Matignon & Tecante, 2017). The 551
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essentially linear polysaccharide with a smaller molecular weight (∼105–6) (BeMiller, 2018). The weight-average molecular weight (Mw) of LS and LS-hydrocolloid blends are presented in Table 1. The results showed that the Mw of LS decreased from 1.283 × 107 ± 6.32% to 1.049 × 107 ± 1.72% g/mol after autoclaving. This might due to the leaching starch molecules (especially amylose) by the autoclave treatment, resulting in a lower average Mw of LS (Ovando-Martínez, Whitney, Reuhs, Doehlert, & Simsek, 2013; Zhang et al., 2014). With the addition of CS, the Mw of LS decreased to 3.251 × 106–7.798 × 105 g/mol (p < 0.05). This might indicating that the more starch molecules leached in LS/CS system, allowing them to orderly align and aggregate more easily, which is likely related to its increased retrogradation phenomenon. Additionally, the Mw of LS decreased as the CS concentration increased, indicating that, theoretically, the degree of retrogradation for 6% LS-0.60% CS should be the greatest among the blends. However, the degree of retrogradation for 6% LS-0.60% CS was lower than those of 6% LS-0.20% CS and 6% LS-0.40% CS based on the G'(t)/G'(0) values and NMR data. This inconsistency might be caused by the increased viscosity in the system when the CS concentrations were greater, based on the pasting viscosity behavior of starch/chitosan composites (Chang, Jian, Yu, & Ma, 2010). The increased viscosity can inhibit the interactions between starch molecules, thus retarding the recrystallization and retrogradation of starch (Shi & Bemiller, 2002). With the addition of GG and XN, the Mw values of LS increased significantly (p < 0.05), which might result from the formation of intermolecular associations between starch molecules and these hydrocolloids. According to previous reports, XN and GG may form intermolecular associations with leached amylose molecules during pasting, which relates to their abilities to prevent phase separation and/ or retrogradation (Fu & Bemiller, 2017; Shi & Bemiller, 2002). Furthermore, the Mw values of LS-XN blends were much greater than those of LS-GG, which might contribute to their lower retrogradation levels. 3.5. Leached amylose of LS-hydrocolloid blends following an autoclave treatment Lotus starch contains a high amount of amylose (∼40%) (Zhang et al., 2015). Generally, amylose will leach out of gelatinized starch dispersions during thermal treatments. The leached amylose aggregates and forms a three-dimensional network embedded with swollen starch granules, which plays an important role in the further gelation and retrogradation of starch (Mariotti, Caccialanza, Cappa, & Lucisano, 2018). The leaching amylose levels of LS and LS-hydrocolloid blends are shown in Table 1. Following autoclaving, the leaching amylose content of LS increased 24.68%–46.90% with CS addition, and decreased with GG or XN addition, especially with the addition of 0.60% XN (decrease of ∼20%). Moreover, an increase in the CS concentration contributed to a greater leaching amylose content (p < 0.05), suggesting that the leaching of amylose from LS could be promoted by the addition of CS. With the addition of GG, the leaching amylose content of LS decreased but no significant difference was found among the different GG concentrations (p > 0.05). In the LS/XN system, until the addition of 0.40% XN (and up), the leached amylose content of LS significantly decreased with the addition of increasing XN concentrations compared with LS (p < 0.05). The diminished molecular weight and greater leached amylose content in the LS/CS system resulted in a greater flexibility, which led to increased reaggregation and rearrangements, while the increased Mw and lower leached amylose content hindered the contact associations between starch molecules in the LS/GG and LS/XN systems, which affected the retrogradation of starch (Kim & Yoo, 2006; Mariotti et al., 2018). Compared with LS, the leached amylose content of LS-XN was significantly decreased until the addition of 0.40% XN (and up). At the 0.40% concentration, significant differences were found in the leached amylose contents of LS-CS, LS-XN and LS-GG. Therefore, each LS/0.40% hydrocolloid system was used to observe the microstructures of
Fig. 3. 13C CP/MAS NMR spectra of native LS, retrograded LS and LS-hydrocolloid blends following autoclaving.
proportion of the amorphous phase of LS decreased with the addition of CS, and increased with the addition of GG or XN. This suggested that the addition of CS facilitated the formation of an ordered structure and promoted starch retrogradation, while that addition of GG or XN resulted in the opposite trends. These findings were consistent with the results of the rheological property analysis. However, a linear relationship between the concentration of each hydrocolloid and its ordered structure was not found; therefore, further studies were needed. 3.4. Mw of LS-hydrocolloid blends following an autoclave treatment Most starch granules are is composed of a mixture of two molecules: amylose and amylopectin. Amylopectin molecules are highly branched with average molecular weights greater than 107, whereas amylose an 552
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Table 1 Chemical shifts, relative proportions of the C4 peaks, Mw values and leached amylose contents of retrograded LS and LS-hydrocolloid blends following an autoclave treatment.
Native LS
LS 6% LS-0.20% CS 6% LS-0.40% CS 6% LS-0.60% CS 6% LS-0.20% GG 6% LS-0.40% GG 6% LS-0.60% GG 6% LS-0.20% XN 6% LS-0.40% XN 6% LS-0.60% XN
C1
C4
C2, 3, 5
C6
Relative proportions of C4 peak (%)
Mw (g/mol)
Leached amylose content (%)
101.60 100.35 99.6 103.04 99.94 103.17 99.83 103.04 98.21 103.13 99.89 103.16 99.99 102.83 100.23 102.83 100.31 103.23 99.8 103.02 102.64 102.99 97.75
82.45
72.75
61.82
4.66
1.283 × 107 ± 6.32%g
–
82.56
72.76
61.91
7.45
1.049 × 107 ± 1.72%h
27.10 ± 0.17d
82.56
72.82
61.84
6.86
3.251 × 106 ± 1.44%i
33.79 ± 0.80c
82.70
72.77
61.91
6.94
8.928 × 105 ± 1.60%j
37.73 ± 0.33b
82.51
72.79
61.92
7.29
7.798 × 105 ± 1.54%k
39.81 ± 0.32a
82.54
72.78
61.92
8.40
1.137 × 107 ± 1.82%f
24.88 ± 0.80e
82.54
72.90
61.91
7.93
1.692 × 107 ± 1.96%e
25.82 ± 0.33e
82.27
72.85
61.82
7.93
1.786 × 107 ± 1.73%d
25.83 ± 0.32e
82.57
72.78
61.89
7.74
3.905 × 107 ± 8.27%a
26.96 ± 0.80d
82.44
72.92
61.93
8.54
3.697 × 107 ± 9.84%b
25.79 ± 0.33e
82.48
72.87
61.84
8.84
3.494 × 107 ± 2.30%c
21.47 ± 0.32f
Different superscript letters indicate statistical significances between averages at p < 0.05.
Fig. 4. AFM of leached amylose in LS-hydrocolloid blends following an autoclave treatment. (A) LS without a hydrocolloid; (B) LS-CS blends; (C) LS-GG blends; (B) LS-XN blends. Scan size is 4 × 4 μm.
553
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an oval shape of varying sizes (Fig. 5A). And CG and XN aqueous dispersions showed weaker fluorescence (white circles) than GG aqueous dispersions (Fig. 5B–D). When heated at 85 °C for 5 min, compared with LS alone (Fig. 5E), the swollen starch granules (red circles in Fig. 5) increased and were larger in size than those in the starch dispersion that contained CS (Fig. 5F). By contrast, when LS granules were heated in both GG (Fig. 5H) and XN (Fig. 5G) solutions, a non-uniform layer of gum existed around the surfaces of the granules, and the swelling and leaching of the starch components was inhibited. Zhang et al. (2018) have reported that the presence of both XN and GG resulted in significant decreases in the swelling extent of corn starch, while arabic gum had little or no effect on the swelling of starch granules during heating. The efficient thickening property and formation of the protecting or lubricating “barrier”, in part, explains why GG and XN restricted the swelling of starch granules effectively as discussed above in relation to leached amylose (Qiu et al., 2015; Zhang et al., 2018). Additionally, the swelling behaviors of LS-hydrocolloid blends following autoclaving were probably attributed to the changes in the leached amylose contents, which play important roles in starch retrogradation (Kim & Yoo, 2006; Mariotti et al., 2018). With the autoclave-cooling treatment, as can be seen in Fig. 5I–L, the starch granules lost shape, swelled and gelatinized to form a continuous and glutinous network. The LS-CS gel (Fig. 5J) appeared to be more compact than the LS gel (Fig. 4I), which might be the result of a reinforced interaction force, such as electrostatic interactions, between LS and CS. The compact structure probably induced the significant increase in the G' value and retrogradation rate of LS. The structures of matrices of mixed pastes supplemented independently with the addition of GG and XN (Fig. 5K and L) showed weak networks with obvious cavities (white arrows) and intermolecular cross-links or association
leaching amylose by AFM (Fig. 4). When compared with LS alone (Fig. 4A), the leached amylose content of the LS/CS system (Fig. 4B) was much smaller (white arrow) and an increasing tendency for particle aggregation (red arrow) occurred following autoclaving. As discussed above, the increased number of leached amylose and amylopectin molecules resulted in the increasing molecular alignment and aggregation in the LS/CS system (Mutungi et al., 2011; Wang et al., 2015). By contrast, the leached amylose contents of the LS/GG and LS/XN systems (Fig. 4C and D) decreased. These structural findings of the AFM correlated with the measurements of leached amylose. Therefore, it was hypothesized that the leaching and aggregation of amylose were affected by different hydrocolloids. The results were consistent with those of previous studies, in which decreases in starch polymer leaching occurred in starch supplemented with GG, XN, corn fiber gum and other hydrocolloids (Qiu et al., 2015; Zhang et al., 2018). The viscosity of the continuous phase may be increased by the presence of hydrocolloids, which prevent the diffusion of starch polymers from starch granules (Funami et al., 2005). Additionally, hydrocolloids, such as XN, are capable of enwrapping the starch granules and physically stabilizing the particle by acting as a protecting or lubricating “barrier”, thus limiting the amount of leached starch polymer (Heyman, Depypere, Meeren, & Dewettinck, 2013). These results were corroborated by the Mw values. 3.6. CLSM of LS-hydrocolloid blends The 6% LS in the absence and presence of each hydrocolloid at 0.40% was selected to observe the swelling process of starch granules during heating and the structure of gelatinized granules following autoclaving using CLSM. Images are displayed in Fig. 5. Native LS showed
Fig. 5. CLSM images of LS and 6% LS-0.40% hydrocolloid blends. (A) native LS; (B) (B) CS; (C) GG; (D) XN; (E) LS without a hydrocolloid heated to 85 °C for 5 min; (F) LS-CS blends heated to 85 °C for 5 min; (G) LS-GG blends heated to 85 °C for 5 min; (H) LS-XN blends heated to 85 °C for 5 min; (I) LS without hydrocolloid gelatinized at 4 °C for 10 h following an autoclave treatment; (J) LS-CS blends gelatinized at 4 °C for 10 h following an autoclave treatment; (K) LS-GG blends gelatinized at 4 °C for 10 h following an autoclave treatment; (L) LS-XN blends gelatinized at 4 °C for 10 h following an autoclave treatment. 554
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(red arrows). Such networks appeared to trap more water molecules in cavities and increase the water-holding capacity of the starch gel, which resulted in lower rates of G' and starch retrogradation.
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4. Conclusions The molecular structures and retrogradation behaviors of LS blended with different hydrocolloids following autoclaving were investigated. The G'(t)/G'(0) values suggested that the retrogradation of LS following an autoclave treatment could be promoted by the addition of CS and retarded by the addition of GG or XN. Based on the changes in G' after the separate addition of NaCl and urea, the electrostatic interactions were the main forces affecting the LS and LS-CS gel, but they were not involved in the formation of LS-GG and LS-XN blends. This indicated that different interaction forces might be underlying factors that affect the properties of LS blended with different hydrocolloids. Compared with LS alone, the lower Mw and increased leached amylose content in the LS-CS blends resulted in a greater tendency to form higher ordered structures and the large aggregates of particles as observed by NMR and AFM, respectively. However, LS-GG and LS-XN showed the opposite trends, and the greater Mw of LS-XN was mainly attributed to its lower retrogradation rate. Furthermore, the enwrapping effects of GG and XN on LS, and the weakened structures for gelatinized LS blends as observed by CLSM, were also attributed to their decreased retrogradation behaviors. These data may have useful applications in producing high-amylose starch/hydrocolloid blends in foods, especially under autoclaving conditions. Declaration of Competing Interest None. Acknowledgements This study was financially supported by the Project of International Cooperation and Exchanges in Science and Technology of Fujian Agriculture and Forestry University (Grant number KXGH17001), the Scientific Research Foundation of Graduate School of Fujian Agriculture and Forestry University (Grant number YB2018009), the National Natural Science Funds (Grant number 31701552), the Leading Talents Support Program of Science and Technology Innovation in Fujian Province (Grant number KRC16002A), the Support Project for Distinguished Young Scholars of Fujian Agriculture and Forestry University (Grant number xjq201714), and the Science and Technology Innovation Project of Fujian Agriculture and Forestry University (Grant number CXZX2017414). References Ambigaipalan, P., Hoover, R., Donner, E., & Liu, Q. (2013). Retrogradation characteristics of pulse starches. Food Research International, 54(1), 203–212. BeMiller, J. N. (2018). Starches: Molecular and granular structures and properties. In J. N. BeMiller (Ed.). Carbohydrate chemistry for food scientists (pp. 159–189). (3rd ed.). Elsevier Inc. Bhat, R., & Sridhar, K. R. (2008). Nutritional quality evaluation of electron beam-irradiated lotus (Nelumbo nucifera) seeds. Food Chemistry, 107(1), 174–184. Cai, X., Hong, Y., Gu, Z., & Zhang, Y. (2011). The effect of electrostatic interactions on pasting properties of potato starch/xanthan gum combinations. Food Research International, 44(9), 3079–3086. Chang, P. R., Jian, R., Yu, J., & Ma, X. (2010). Fabrication and characterisation of chitosan nanoparticles/plasticised-starch composites. Food Chemistry, 120(3), 736–740. Chen, L., Ren, F., Zhang, Z., Tong, Q., & Rashed, M. M. A. (2015). Effect of pullulan on the short-term and long-term retrogradation of rice starch. Carbohydrate Polymers, 115, 415–421. Fu, Z., & Bemiller, J. N. (2017). Effect of hydrocolloids and salts on retrogradation of native and modified maize starch. Food Hydrocolloids, 69, 36–48. Funami, T., Kataoka, Y., Omoto, T., Goto, Y., Asai, I., & Nishinari, K. (2005). Effects of non-ionic polysaccharides on the gelatinization and retrogradation behavior of wheat starch. Food Hydrocolloids, 19(1), 1–13.
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