Inhibition of gelatinized rice starch retrogradation by rice bran protein hydrolysates

Accepted Manuscript Title: Inhibition of gelatinized rice starch retrogradation by rice bran protein hydrolysates Authors: Liya Niu, Leiyan Wu, Jianhui Xiao PII: DOI: Reference:

S0144-8617(17)30846-9 http://dx.doi.org/doi:10.1016/j.carbpol.2017.07.070 CARP 12587

To appear in: Received date: Revised date: Accepted date:

12-5-2017 17-7-2017 24-7-2017

Please cite this article as: Niu, Liya., Wu, Leiyan., & Xiao, Jianhui., Inhibition of gelatinized rice starch retrogradation by rice bran protein hydrolysates.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2017.07.070 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Inhibition of gelatinized rice starch retrogradation by rice bran protein hydrolysates Liya Niu, Leiyan Wu and Jianhui Xiao*,a, b

a

School of Food Science and Engineering, Jiangxi Agricultural University, 1101 Zhimin Road,

Nanchang 330045, P.R. China b

Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education,

Jiangxi Agricultural University, Nanchang, China

*

Corresponding author: [email protected]

Telephone: +86-15083838985

Highlights 

Protamex-hydrolyzed rice bran protein at 1 h (PRBPH-1) was released.



PRBPH-1 reduced significantly the short- and long-term retrogradation of gelatinized rice starch.



The relative crystallinity of retrograded RS declined with the addition of PRBPH-1 in DSC and XRD.



Formation of intra- and inter-particle hydrogen bonds and recrystallization were weakened by PRBPH-1.

Abstract The retrogradation of gelatinized rice starch (GRS) during the shelf life of a product is 1

the biggest barrier related to starch-containing foods. The objective of this study was to produce rice bran protein hydrolysate (RBPH) using proteolytic enzymes (alcalase, flavourzyme, protamex, neutrase, bromelain, papain and trypsin) to suppress the retrogradation of GRS and understand the physical phenomena underlying the reduced retrogradation of GRS by RBPH during short- and long-term storage. Mixtures of GRS incorporated with Protamex-hydrolyzed rice bran protein at 1 h (PRBPH-1) at a degree of hydrolysis of 15.1% were still fresh after storage at 4 °C for 14 d. The dynamic time sweep results obtained at 4 °C for 180 min showed that PRBPH-1 reduced the storage modulus to a greater extent, indicating that PRBPH-1 suppressed the short-term retrogradation of GRS. Differential scanning calorimetry (DSC) clearly showed that PRBPH-1 significantly decreased the retrogradation enthalpy during the 28-d storage at 4 °C, and the retrogradation kinetics were analyzed by the Avrami model. In addition, the recrystallization of GRS based on X-ray diffraction spectroscopy was reduced from 15.41% to 4.86% when the GRS: PRBPH-1mass ratio increased from 100:0 to 100:12. Confocal laser scanning microscopy, atomic force microscopy and scanning electron microscopy demonstrated that PRBPH-1 dispersed between GRS molecules to block the formation of hydrogen bonds to inhibit the recrystallization of GRS. These findings suggested that PRBPH-1 inhibited the short- and long-term retrogradation of GRS, and can be potently employed as a natural alternatives for improving the quality and nutrition of starch-containing foods.

Keywords: Rice bran protein, enzymatic hydrolysis, rice starch, retrogradation, recrystallization, structure

1. Introduction Steamed rice, as well as rice noodles, rice cakes, instant rice, and rice pasta, is a staple 2

food for over half of the world’s population, especially in Asian countries such as China, Japan, Thailand, Malaysia, India, and Sri Lanka (Ahmed, Qazi, Li, & Ullah, 2016; Chung, Rico, Lee, & Kang, 2016; Tsuiki, Fujisawa, Itoh, Sato, & Fujita, 2016; L. Wang, et al., 2016). However, due to the retrogradation of gelatinized rice starch (GRS), the fresh, soft, pliable and elastic rice products decrease water retention capacity to become tough, rigid and fragmentation, as well as opacity. As a consequence, these changes can severely deteriorate the quality and shorten storage stability of starch-containing gel products, resulting in the food wastage and economic losses (Lian, Kang, Sun, Liu, & Li, 2015; Tian, et al., 2013; Yanjun Zhang, et al., 2014). Many coexisting substances, such as proteins, carbohydrates, lipids, acids, salts, polyalcohols, polyphenols, emulsifiers, and amylase, have shown the ability to reduce the short- and long-term retrogradation of GRS to different extents (Fu, Chen, Luo, Liu, & Liu, 2015; Shujun Wang, Li, Copeland, Niu, & Wang, 2015). Recently, fast-paced lifestyle and seeking for convenience have contributed to the evolution of novel food trends to develop products that meet the created demand (Alauddina, Islama, Shirakawaa, Kosekib, & Komaia, 2017; K Boonloh, et al., 2015; Kampeebhorn Boonloh, et al., 2015). Foods with clean label, freeform and natural functional ingredients, can satisfy consumer demands (Graves, et al., 2016). Starch-containing gel products incorporated functional ingredients with health-benefitting properties, can be potential to elicit the health benefits when introduced into consumer’s diet. As a consequence, there has been a great deal of interest in finding the appropriately natural alternatives to coexist with starch to inhibit the retrogradation of GRS and meet the consumers’ proactive demand. In recent years, protein hydrolysates (RBPHs) isolated from rice bran have been

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proposed for special nutritional and biological properties, such as antioxidant, antiproliferation, anticarcinogenic and antiallergic activities (Alauddina, Islama, Shirakawaa, Kosekib, & Komaia, 2017; K Boonloh, et al., 2015; Boonloh, Kukongviriyapan, Kongyingyoes, Kukongviriyapan, Thawornchinsombut, & Pannangpetch, 2015). Based on the health-promotion properties of RBPHs, it can serve as economical and natural alternatives that can be used in the food system to satisfy consumer demands. There is no doubt that blending RBPHs directly to the powdered starch can become part of system. When gelatinized, they are prone to interact with each other. However, information about the multiple effects of RBPHs on the overall product quality is few, motivating research in this area (Graves, Hettiarachchy, Rayaprolu, Li, Horax, & Seo, 2016). If adding RBPH to rice starch-containing foods can retard the retrogradation of GRS, RBPH may be used to improve the nutrition and quality of rice starch-based products. Hence, the objective of our study was to prepare RBPH to prevent the retrogradation of GRS using a food-grade proteolytic enzyme. Further, dynamic rheology, differential scanning calorimetry (DSC), X-ray diffraction (XRD) spectroscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), as well as atomic force microscopy (AFM) were be used to interpret the short- and long-term retrogradation properties with respect to the structures. 2. Materials and methods 2.1 Materials Rice starch with an amylose content of 23.2% and rice bran were purchased from Golden Agriculture Biotech Co., Ltd. (Shanggao, Yichun, China). Freeze-dried rice bran

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protein (RBP) was prepared using the method described in our earlier publication (Xiao, Zhang, & Niu, 2013). Nile blue was from Sigma-Aldrich Corp. Alcalase FG (2.4 AU/g), flavourzyme 500 G (500 LAPU/g), protamex (1.5 AU/g) and neutrase 1.5 MG (1.5 AU/g) were obtained from Novozymes Biotechnology Co., Ltd. (China). Bromelain (1200000 U/g), papain (3000000 U/g) and trypsin (4000 U/g) were purchased from Nanning Pangbo Bioeng Co., Ltd. (Nanning, China). All other chemical solvents used were of analytical grade and purchased from Sinopharm Chemical Reagent Factory (Shanghai, China). 2.2 Methods 2.2.1 Preparation of RBP hydrolysates (RBPHs) RBPHs was prepared using the method described by Xiao et al. (Xiao and Zhang 2012). The RBP was digested for 0, 0.5, 1, 2 and 4 h to various degrees of hydrolysis (DH) with the commercial protease at the optimum pH, the ratio of enzyme to substrate concentration (E/S) and temperature conditions, as shown in Supporting Information (Table 1). The pH of the mixture was maintained at a constant value by adding 3 mol/L HCl or 3 mol/L NaOH during hydrolysis. Thermal treatment at 95 °C for 15 min was used to stop the hydrolysis reaction. Next, the hydrolysates were centrifuged at 4500×g for 15 min, and the supernatant was lyophilized to prepare the RBPH. The DH was calculated using Eq. (1):

 AN 2  AN 1   ×100 DH (%)   N p b  

(1)

where AN 1 and AN 2 are the amino nitrogen content of the protein substrate before and after hydrolysis (mg/g protein), respectively, and Npb is the nitrogen content of the peptide bonds in the protein substrate (mg/g protein). 2.2.2 Measurement of the anti-retrogradation activity of RBPHs

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The hardness was tested in a texture profile analysis (TPA) to evaluate the anti-retrogradation activity of RBPHs. RS-RBPH mixtures with mass ratios of 100:6 were prepared. In brief, 10 mL of deionized water and 5 g of RS-RBPH mixtures were mixed in a 25 mL beaker for 5 min using a vortex-5 mixer (Haimen Kylin-Bell Lab Instruments Co., Ltd.), followed by heating at 90 °C for 30 min in a water bath (SKY-111B, Shanghai Sukun Industry and Commerce Co., Ltd.) and cooling for 1 h at ambient conditions. The gelatinized samples (at 0 d and after storage at 4 °C for 14 d) were compressed twice using a cylindrical probe with a diameter of 7 mm at a speed of 1 mm/s and a force of 10 g. The deformation level was 30% of the original sample height. Each treatment was tested in triplicate. 2.2.3 Dynamic viscoelastic measurements Dynamic viscoelastic measurements were carried out using a cone-plate geometry (AR-G2 rheometer, TA Instruments Inc., USA) configured with a hard anodized split solvent trap cover to reduce solvent evaporation. According to the TPA results, the Protamex-hydrolyzed rice bran protein at 1 h (PRBPH-1) showed the highest anti-retrogradation activity that was used in the following tests. PRBPH-1 was mixed with RS at mass ratios of 100:0, 100:3, 100:6, 100:9, and 100:12. Then, the suspensions were prepared by mixing 5 g of the RS-PRBPH-1 mixtures with 10 mL of deionized water for 5 min at room temperature using a vortex-5 mixer, and the mixtures were transferred to a water bath and heated at 90 °C for 30 min. The slurries obtained from the water bath were immediately transferred to the rheometer plate. After locating the cone, the excess sample was removed. Prior to measurement, the slurries were cooled to 4 °C and held for 10 min to equilibrate stress and temperature. Changes in the storage moduli (G) and the tan δ of the isothermal

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time sweep of 3 h at 4 °C were monitored during aging at a constant frequency of 1 Hz and a constant strain of 1%, which was within the limit of the linear viscoelastic region for all samples (data not shown) (Tang, Hong, Gu, Zhang, & Cai, 2013). 2.2.4 Differential scanning calorimetry (DSC) The onset temperature (To), peak temperature (Tp), conclusion temperature (Tc), and enthalpy (H) of rice starch (RS) with different PRBPH-1 ratios (100:0, 100:3, 100:6, 100:9, or 100:12) was monitored by DSC (DSC 214 Polyma, NETZSCH-Gerätebau GmbH, Germany). Prior to measurement, the instrument was calibrated using indium as a standard. In detail, an RS-PRBPH-1 mixture (5.0–5.2 mg) was weighed into an aluminum pan, and deionized water was added using a Finnpipette (Thermo Fisher Scientific, Shanghai, China) to give a water-to-mixture (dry solid) ratio of 2:1 (w/w). The sealed samples were equilibrated for 12 h at 4 °C and then heated from 25 to 110 °C at 5 °C/min to determine H0. After the first-run heating, the gelatinized samples were cooled and stored at 4 °C for 1, 3, 5, 7, 14, 21 and 28 d (H. Zhang, Sun, Zhang, Zhu, & Tian, 2015). The stored samples were heated again at the same heating rate over the same temperature range to determine Ht. An aluminum pan containing 10 μL of distilled water served as a reference. The Avrami equation (Eq. (2)), which describes crystallization at temperatures above the glassy region, has been extensively used by many researchers to illustrate the retrogradation kinetics of starch (especially amylopectin) to provide a convenient empirical means of representing the process of starch retrogradation. ∆𝐻 −∆𝐻

X(t)=∆𝐻 𝑡 −∆𝐻0 =1-exp(-ktn) ∞

0

(2)

where X(t) and Ht are the fraction of crystallized starch and the enthalpy change,

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respectively, at 1, 3, 5, 7, 14, and 21 d; H∞ is limiting enthalpy change (28 d for all samples); and k and n are the rate constant and Avrami exponent, respectively 2.2.5 X-ray diffraction (XRD) spectroscopy XRD analysis was performed for treatments with RS:PRBPH-1 mass ratios of 100:0, 100:6, and 100:12. The heated samples were prepared as in the TPA analysis and stored at 4 °C for 14 d. Prior to XRD analysis, the samples were freeze-dried and milled to pass through a 60-mesh sieve. XRD analysis was performed using a D8 ADVANCE-A25 X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany). Scanning was performed with a voltage of 30 kV, current of 10 mA, wavelength of 0.154 nm, and diffraction angle (2θ) between 4 and 40° at a rate of 2°/min. The rice starch was used as a control. Jade version 6.5 software was used to analyze the diffractograms, and the curved-line mapping method was used to calculate the relative crystallinity (Yachuan Znhang & Rempel, 2012). 2.2.6 Confocal laser scanning microscopy (CLSM) Treatments with RS:PRBPH-1 mass ratios of 100:0, 100:6, and 100:12 were characterized using a Leica TCS SP8 microscope (Leica Microsystems, Heidelberg GmbH, Germany). To prepare samples for CLSM, different amounts (0, 6, and 12 mg) of PRBPH-1 were dissolved in 10 mL of deionized water, and Nile blue was added at 3% mass of PRBPH-1. After the mixture was gently stirred for 4 h at 21 °C, in order to remove the contaminants such as the extra Nile blue and salts, PD MiniTrapTM G-10 columns (GE Healthcare Bio-Sciences Corp., 800 Centennial Avenue, P.O. Box 1327, Piscataway, NJ 08855-1327, USA) were used for cleanup of the mixture of PRBPH-1 and Nile blue. And this experiment procedure was repeated six times. After the cleanup step, 100 mg of RS was

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added to the above mixtures to obtain the targeted RS-PRBPH-1. Then incubation for 30 min at 90 °C in a water bath and cooling for 1 h at ambient temperature (21 °C), 5 μL of a stained RS-PRBPH-1 mixture was dropped onto a glass slide and covered with a piece of cover glass, followed by imaging (Yun Zhang, Lin, & Zhong, 2015). 2.2.7 Atomic force microscopy (AFM) A MultiMode 8 microscope (Bruker Corp., Santa Barbara, CA) was used to study the structures of treatments with RS:PRBPH-1 mass ratios of 100:0, 100:6, and 100:12. To prepare the samples for AFM, 0.1 g of an RS-PRBPH-1 mixture was dissolved in 10 mL of deionized water, heated at 90 °C for 30 min in a water bath, and cooled for 1 h at ambient conditions (21 °C). After diluting to 0.1 mg/mL of the solutes in deionized water, 5 µL of the diluted mixture was spread evenly onto a freshly cleaved mica sheet. After vacuum drying for 15 min at 45 °C, the samples were scanned using a rectangular cantilever probe, and the topographic images were characrized in the SCANASYST-AIR mode (Guan & Zhong, 2015). 2.2.8 Scanning electron microscopy (SEM) First, the RS-PRBPH-1 mixtures (0.5 g) with mass ratios of 100:0, 100:6, and 100:12 were dissolved in 1 mL of deionized water, then heated at 90 °C for 30 min and cooled for 1 h to room temperature. The cooled mixtures were placed in an ultra-low temperature refrigerator at -60 °C for 24 h and then freeze-dried (Scientz-10N freeze dryer, Scientz Co., Ltd, Ningbo, China) at 0.1 mbar and -75 °C for 24 h. The fractured middle part of the cross-sectional area of the freeze-dried spongy material was mounted onto a stub using double-sided conductive adhesive tape and subsequently coated with gold prior to SEM (FEI

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Quanta 250, FEI, USA) observation at 10 kV. 2.2.9 Statistical analysis All data were analyzed using SPSS 20.0 statistical software (IBM, USA). The mean and standard deviation (SD) were calculated from three replicates (n = 3). The least significant difference (LSD) method was used to analyze the differences among treatments at a significance level of 0.05. 3. Results and discussion 3.1 Effects of RBPHs on the retrogradation of GRS. As shown in previous studies (Fu, Chen, Luo, Liu, & Liu, 2015; H. Zhang, Sun, Zhang, Zhu, & Tian, 2015), the retrogradation of GRS is associated with an increase in the firmness of starch-based products. Therefore, the hardness of starch gel is quite sensitive and highly correlated to the retrogradation of starch and can be used as an index of starch retrogradation. The variation in the hardness of the sample gels incorporated with RBP and its hydrolysates (RBPHs) stored at 4 °C for 0 and 14 d was shown in Fig. 1. The RBPHs prepared by using seven commercial proteases (alcalase, flavourzyme, protamex, neutrase, bromelain, papain and trypsin), resulted in significant differences in the hardness, but with no discernible regularity. In other words, not all RBPHs can inhibit rice starch retrogradation. Compared to rice starch and RS-RBP mixture, rice starch blended with hydrolysates obtained at different hydrolysis times (0.5, 1, 2, and 4 h) by the seven proteases showed significantly lower hardness. In contrast to the other six proteases, hydrolysates prepared by Protamex produced much lower hardness. And it was important to point out that Protamex-hydrolyzed rice bran protein at 1 h (PRBPH-1) resulted the lowest hardness (117.52 g) and starch gel was fresh

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after storage at 4 °C for 14 d, indicating that PRBPH-1 equiped with the highest anti-retrogradation activity and thus was selected for further study. In order to explicate this phenomenon, the basic structure of PRBPH-1 was also studied, which composed of 10.76%, 70.28%, and 18.96% for molecular mass of >1000, 180-1000, and <180 Da according to size exclusion chromatography. And the charge density and hydrophobicity of PRBPH-1 at pH 7.0 were 0.684 and 5.21 kJ/mol, respectively. According to reports in the literature, Xijun Lian et al (2013) reported that soy protein hydrolysates with various DH showed significant differences in retarding the maize starch retrogradation, due to the different lengths of peptides or degree of hydrolysis. Boyle, Daniel L. et al (2007) noted that proteins hydrolysates significantly softened the sorghum batters gel, and the interpretation was the shorter chain lengths and the end resides of proteins hydrolysates. Qian Liu et al. (2016) suggested that increased porcine plasma protein hydrolysates (PPPH) can decrease gel penetration force. These gel texture results can be explicitly illuminated by the small molecular weight of PPPH adsorbing on the surfaces of starch granules. Min Cui et al. (2014) reported that Arg (alkaline amino acid) and Glu and Asp (acidic amino acids) decreased the gel strength of potato starch depending on the absolute value of its net charge. While neutral amino acids (Phe and Met) can not cause modifications in starch gels. As discussed previously, a common interpretation is that the chain lengths, charge density and amino acid sequences of peptides in course of hydrolysis (In Supporting Information, as shown in Fig. 1) weaken the interactions between amylose and amylopectin and therefore short- and long-range structures in starch gels to reduce hardness. Our conclusion also herein confirms this explanation.

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3.2 Effect of PRBPH-1 on the short-term retrogradation of RS Previous literatures (Fu, Chen, Luo, Liu, & Liu, 2015) confirmed that starch retrogradation consists of short- and long- term. Amylose reassociates to an ordered structure and is thought to be the fraction responsible for short-trem retrogradation in early stages, determined by rheological methods. While the slower and thermically recrystallization of amylopectin can be clarified to the long-term retrogradation. As shown in Fig. 2A, in the initial stage of the measurement, addition of PRBPH-1 reduced the value of G. And The G values of the rice starch gels prepared with PRBPH-1increased slowly and were lower than that of the rice starch paste throughout the experiment. Moreover, the G values of the rice starch/ PRBPH-1 system declined when the amount of PRBPH-1 was increased. Additionally, the changes in the dynamic mechanical loss tangent (tan δ) during storage were investigated and illustrated in Fig. 2B. The tan δ values increased with the increase in PRBPH-1 concentration but decreased with the extension of the storage time. These results indicated that retrogradation had occurred in all the samples. The mixtures that contained PRBPH-1 had higher tan δ values than starch alone did, which suggested that the gelation behavior of rice starch was affected by PRBPH-1.These data showed that PRBPH-1 can weaken the three dimensional gel network structure. Therefore, adding PRBPH-1 to rice starch system clearly suppressed the short-term retrogradation of rice starch, which may be explained by the fact that PRBPH-1 can inhibit the dissolution of amylose during the pasting process. Tsai, ML. et al. (Tsai, Li, & Lif, 1997) noted that amylose acts as both a diluent and an inhibitor of swelling that results the rigid starch gel. This supports our finding that the G peak decreased with the decrease in amylose content in the rice starch suspension. Our interpretation in the

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present study was in agreement with the previous report that suggested that Arg probably restrained amylose leaching from potato starch granules, resulting in a decreased retrogradation tendency (W. Chen, Zhou, Yang, & Cui, 2015). 3.3 DSC analysis The gelatinization properties of RS-PRBPH-1 mixtures determined by DSC were summarized in Table 1. With the increase in the content of PRBPH-1, the onset (To) and peak (Tp) temperatures of the rice starch noticeably increased from 64.8 °C to 69.4 °C and 71.1 °C to 76.2 °C, respectively. Similar gelatinization properties of starch suspensions have been reported previously. Qian Liu et al. (Kong, Niu, Sun, Han, & Liu, 2016) suggested that increased porcine plasma protein hydrolysates can significantly enhanced the gelatinization temperatures of corn starch. This increasement is likely due to the replacement with proteins that can additionally retained water to the starch granules. It is well-known that pasting occurs when the starch granules absorb sufficient water and swell after gelatinization (Yachuan Zhang & Rempel, 2012). The previous reports (L. Chen, Ren, Zhang, Tong, & Rashed, 2015) (Fu, Chen, Luo, Liu, & Liu, 2015) showed that adding the proteins to starch suspension can change in the overall net charge of the rice starch suspension, resulting in more space and more repulsion forces for water molecules between the proteins. This repulsion forces increase with increasing charge, resulting that more water molecules can be attached to the protein strands. Therefore, less mobile water is available in the rice starch system. This results in an increased pasting temperature which is in according with the interpretation reported by Ito et al. (2006). While PRBPH-1 decreased the conclusion temperatures (Tc) and enthalpy change ( △ H) from

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82.6 °C to 78.2 °C and 11.46 J/g to 7.44 J/g, respectively. We speculated that this phenomenon is probably attributed to the strong alkali environment produced by hydroxide ions in PRBPH-1, which make RS more un-stable than the control. Min Cui et al. (2014) noted that the adding alkaline amino acid to potato starch significantly decreased the Tc, due to its hydroxide ions. The H changes of the rice starch and RS-PRBPH-1 mixtures during storage at 4 °C was shown in Table 2. H increased as the number of storage days increased, indicating that a large number of crystallites formed during storage.Therefore, more energy was required to melt these crystallites (Fu, Chen, Luo, Liu, & Liu, 2015). As the storage time increased from 0 to 28 d, the H of RS increased significantly from 0 to 7.12 J/g. Compared with RS, the H values decreased with the addition of PRBPH-1, and lower H values were obtained when greater amounts of PRBPH-1 were added. The enthalpies of the RS-PRBPH-1 mixtures decreased from 7.12 to 2.36 J/g as the PRBPH-1 ratios increased from 100:0 to 100:12 when stored for 28 d. These results suggested that PRBPH-1 could inhibit the long-term retrogradation of RS, especially the crystallization of amylopectin (Shujun Wang, Li, Copeland, Niu, & Wang, 2015). In previous studies, the recrystallization kinetics of starch were shown to fit the Avrami equation (Xu, et al., 2013). The Avrami exponent (n) and rate constant (k) of the Avrami model are shown in Table 2. The high determination coefficient values (R2, 0.9471–0.9919) approached 1, indicating that the Avrami theory fit well with the recrystallization kinetics of our RS-PRBPH-1 mixtures. Previous studies have reported that the parameter k depends on the crystal growth and crystal nucleation constants. While the parameter n depends on the

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nature of crystal nucleation and the crystal dimensions (S. Wang & Copeland, 2012). Compared with the RS alone, RS-PRBPH-1 had a slower recrystallization rate (k, 0.375-0.149), while the Avrami exponent (n) of RS-PRBPH-1 (0.934) was higher than that of RS (0.576). These results might have been due to the active polyhydroxy groups in PRBPH-1, which may block or be inserted into the hydrogen bonds among the starch molecules (H. Zhang, Sun, Zhang, Zhu, & Tian, 2015). Subsequently, the transformation of the nuclei changed the environment of the starch molecules. These results suggested that adding PRBPH-1 can slow down the recrystallization of rice starch. Therefore, PRBPH-1 could be used to prolong the shelf life of foods containing starch. 3.4 XRD analysis As discussed in DSC, the long-term retrogradation increases the crystallinity of starch gels, resulting in the increased height and decreased width of XRD diffraction peaks (Shujun Wang, Li, Copeland, Niu, & Wang, 2015). Fig. 3 illustrated the XRD diffractograms of preheated RS-PRBPH-1 mixtures after storage at 4 °C for 14 days. The diffractogram of pristine rice starch exhibited characteristic peaks at 2 angles of 15.0°, 17.0°, 17.9°, and 22.8°, which are typical of A-type peaks (L. Chen, Ren, Zhang, Tong, & Rashed, 2015; Soares, et al., 2011; Wu, Che, & Chen, 2014), and the degree of recrystallinity was 21.62%. However, as the storage time increased, the retrogradation of GRS was evident from the appearance of a B-type pattern at 2 angles of 16.9° and 20.8° after storage for 14 d. The different 2 angles of the diffraction peaks observed for pristine RS and GRS are correlated with the transformation from A- to B-type crystalline structures (L. Chen, Ren, Zhang, Tong, & Rashed, 2015; Li, et al., 2013). With the increased mass of PRBPH-1 in the mixture, the

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degree of recrystallinity reduced from 15.41% to 4.86% during the 14-d storage period, indicating that adding PRBPH-1 to RS could prevent the recrystallization of amylose and/or amylopectin to enable the reduction of retrogradation supposed by the DSC results. 3.5 Morphology of the RS-PRBPH-1 mixtures Although rheology, DSC and XRD showed that PRBPH-1 significantly lowered the recrystallization of amylose and/or amylopectin to enable a decreased trend of retrogradation, these studies did not provide a physical basis to interpret the phenomena. Information about particle structures is therefore needed to understand the above results of rheology, DSC and XRD. The mesoscopic structures of the RS-PRBPH-1 mixtures were observed by CLSM (Fig. 4) after heating at 90 °C for 30 min and cooling to 21 °C. With the increase mass of PRBPH-1, PRBPH-1 with more staining was more distributed in the peripheral layer of the rice starch (Fig. 4B). No separate regimes of blue fluorescence (PRBPH-1) were observed for any of the samples. It can manifest that PRBPH-1 can associate with the aggregated starch structures and granule remnants after heating and cooling. Qian Liu et al. (2016) showed that after cooling, the presence of porcine plasma protein hydrolysates in amylopectin and granule remnants can lower the visible starch granule ghost. Moreover, the penetration of PRBPH-1 into the swollen starch structures and granule remnants during heating to interact with components was showed in the morphology (Fig. 4 C and D). The images indicated that PRBPH-1 impacted the structures of amylopectin and granule remnants after heating and cooling (Fig. 4), not amylose in the continuous phase. After heating and releasing amylose, the disrupted starch granules structures enable the

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interactions between PRBPH-1 and starches, likely due to electrostatic and also possibly hydrophobic attraction with slightly negatively charged amylopectin, as reported by BeMiller et al (BeMiller & Huber, 2010). After cooling, the presence of PRBPH-1 in granule remnants and amylopectin affects the formation of hydrogen bonds within particulates and those with continuous phase amylose to reduce the extent of recrystallization and retrogradation. The results were in agreement with our recent study observed in rheology, DSC, and XRD (Figures 2A and 3, Table 2). And the presence of PRBPH-1 in and on particulates weakens intra- and inter-particle interactions to reduce the solid-like properties of gels, as observed in rheology (Fig. 2). The nanostructure morphology of PRBPH-1, gelatinized RS and the RS-PRBPH-1 mixtures was also observed by AFM, as shown in Fig. 5. Possibly due to much lower concentration of solution and further dilution in AFM sample preparation, the structures in AFM were considerably smaller than those in CLSM (Fig. 4). The images showed PRBPH-1 with globular morphology that composed of small aggregates with 1.8 nm height. The height of PRBPH-1 tested in the present study was in agreement with the study that reported the height of the aggregates of peptides in aqueous solutions to be 1.5-6 nm (Enache, Chiorcea-Paquim, & Oliveira-Brett, 2016). However, the GRS condensed to irregular structures about 3.4nm height and extensive deposits. In the previous studies, using the drop deposition method that heated at 95 °C and drying at 70 °C on mica sheets, Maley et al. (Maley, et al., 2010) noted that barley starch with 0, 25.8, and 38% amylose appeared mainly as small particles and aggregated globules heightened form 1.8 to 5.5 nm. Hongjie An et al. reported that potato starches formed networks with the height from 0.3 to 11.0 nm under

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microwave radiation (An, Yang, Liu, & Zhang, 2008). The impact of amylose content and heating modes on the nanostructures of gelatinized starch was studied by Maley et al. (Maley, Asare, Båga, Rossnagel, Chibbar, & Sammynaiken, 2010) and Hongjie An et al. (An, Yang, Liu, & Zhang, 2008). Rice starch in the present study had 23.2% amylose that heated at 90 °C for 30 min in a water bath. Therefore, the irregular structures observed in AFM with a height of a few nm (Fig. 5B) agreed with the study of Maley et al. (Maley, Asare, Båga, Rossnagel, Chibbar, & Sammynaiken, 2010) and Hongjie An et al. (An, Yang, Liu, & Zhang, 2008). After the addition of PRBPH-1, the nanoparticles of PRBPH-1 scattered on the surface of RS (Fig. 5C). Moreover, with the increasing addition of PRBPH-1 (Fig. 5D), PRBPH-1 densely packed together on the surface with the height of the RS-PRBPH-1 particles to ~ 7 nm, which was approximately 3-5 times that of PRBPH-1. Therefore, it can be suggested that the aggregation of amylose and amylopectin at the nanoscale was affected by PRBPH-1, likely molecular level. The results were consistent with those of previous studies (Barbiroli, et al., 2013; BeMiller & Huber, 2010). The microstructures of freeze-dried gelatinized RS and RS-PRBPH-1 mixtures was clearly observed via SEM (Fig. 6). Clear microstructural differences could be observed between the gelatinized RS and RS-PRBPH-1 mixtures gels. In the electron micrograph of GRS alone (Fig. 6A1 and A2), GRS formed irregular lamellas, suggesting RS granules was the sufficient gelatinization and thorough disintegration when gelatinized at 90°C for 30 min. This conclusion was supported by the results of DSC (Table 1) that PRBPH-1 can decrease the ended temperature of gelatinization from 82.6 °C to 78.2 °C. What is more, many holes (Fig. 6A2, arrow a1) interspersed on the irregular lamellas. These formed holes were

18

attributed to the water separation and the rearrangement of RS molecules. Previous studies evaluated the sufficient gelatinization and thorough disintegration of RS molecules can form three-dimensional gel networks filled by water, as also observed in our dynamic viscoelastic measurements (Fig.2). During cold storage, the interactions between the starch molecules strengthened and aggregated together through hydrogen bonds, resulting in water gradually draining from the gel networks. When subjected to freeze dehydration, water escaping from the gel networks formed holes on the irregular lamellas (Fu, Chen, Luo, Liu, & Liu, 2015; Shujun Wang, Li, Copeland, Niu, & Wang, 2015).. However, the intact film in the RS-PRBPH-1 mixtures was displayed at the arrow in Fig. 6b2 and c2. Additionally, small cylinder obviously stood on the film, as shown at the arrow in Fig. 7b1 and c1. And the presence of layered structure was observed in Fig. 6B and C. Moreover, greater amount of PRBPH-1 resulted in a bigger gap between the layers. This observation could be interpreted by the fact that the interactions increase among PRBPH-1, leached amylose, amylopectin, granule remnants and water (Baldwin, Adler, Davies, & Melia, 2010; S. Wang & Copeland, 2012). The presence of PRBPH-1 in leached amylose and amylopectin

decreased

the

amount

interactions

of

amylose-amylose

and

amylose-amylopectin to restrict the diffusion and exudation of water, which was beneficial for establish a network-like structure (Ito, Hattori, Yoshida, Watanabe, Sato, & Takahashi, 2006; Kong, Niu, Sun, Han, & Liu, 2016). Meanwhile, PRBPH-1 densely packed together on the surface with the height of the RS-PRBPH-1 particles to ~ 7 nm (Fig.6), accounting for the interesting phenomenon of small cylinder and layered structure. Therefore, the combined results described in CLSM, AFM and SEM indicate that PRBPH-1 bound with GRS starch

19

molecules to block the formation of hydrogen bonds to impact intra- and inter-particle interactions and inhibit the recrystallization of GRS. In addition, the findings in this section also correspond to the rheological properties and DSC and XRD results.

4. Conclusions In the present study, the anti-retrogradation activity of RBP hydrolysates (RBPHs) from alcalase, trypsin, protamex, flavourzyme, papain, neutrase and bromelain were investigated by the TPA assay. Compared to RBP, the anti-retrogradation activities of RBPHs were significantly higher. The Protamex-hydrolyzed rice bran protein at 1 h (PRBPH-1) possessed the highest anti-retrogradation activity. And the gradual addition of PRBPH-1 resulted in a dramatic reduction of storage modulus (G'), indicating that PRBPH-1 weakened the interactions between amylose and amylopectin and therefore short- and long-range structures in starch gels to soften GRS gels. The binding by PRBPH-1 increased the gelatinization temperature of rice starch as observed in DSC. And DSC and XRD results revealed that higher PRBPH-1 content in rice starch reduce the extent of recrystallization and long-term retrogradation, which could be interpreted by the mesoscopic and microcosmic structures of the RS-PRBPH-1 mixtures shown in CLSM, AFM, and SEM that PRBPH-1 bound with granule remnants and amylopectin during rice starch gelatinization and after cooling and subsequently impacted the surface and interior structures of particulates in GRS gels. Our findings suggested that PRBPH-1 might be a potentially natural alternatives for improving the quality and nutrition of starch-containing foods during short- and long-term storage.

20

5. Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 31671896) and General Financial Grant from the China Postdoctoral Science Foundation (2017M612152). And JH. Xiao would like to acknowledge the support from The National Natural Science Foundation of Jiangxi Province (No. 20151BBF60043 and No. 20151BBF60045).

21

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27

1000

A

a

control* 0h 0.5h 1h 2h 4h

Hardness (g)

800 600

b c d e

e

400 200

a

ab

bc bc

d

d

0 0d

14d Storage time

1000

B

a

control* 0h 0.5h 1h 2h 4h

Hardness (g)

800 600

b

b

b

b

b

400 200

a

a

b bc bc

c

0 0d

14d Storage time

1000

C

a control* 0h 0.5h 1h 2h 4h

Hardness (g)

800 600

b

400 c

200

a

c d

b de

e

c

cd

0 0d

14d Storage time

28

d

1000

D

a control* 0h 0.5h 1h 2h 4h

Hardness (g)

800 600

b c

c

c

c

b

b

c

c

400 200

a

a

bc bc

b

c

0 0d

14d Storage time

1000

E

a control* 0h 0.5h 1h 2h 4h

Hardness (g)

800 600

b

b c

400 200

a

b

cd e

c

d

0 0d

14d Storage time

1000

F

a control* 0h 0.5h 1h 2h 4h

Hardness (g)

800 600

b

c

c

400 200

a

a

b

c

c

bc

0 0d

14d Storage time

29

1000

G a control* 0h 0.5h 1h 2h 4h

Hardness (g)

800 600

b c

c

c

c

400 200

a

b

d

d

c

d

0 0d

14d Storage time

Fig.1 Effects of RBP (0 h) and RBPH (0.5, 1, 2 and 4 h) obtained using different enzymes on the retrogradation of gelatinized RS. A, B, C, D, E, F and G correspond to the alcalase, trypsin, protamex, flavourzyme, papain, neutrase and bromelain treatments, respectively. *RS without RBP and RBPH was tested and served as the control.

30

3750

A 100:0 100:3 100:6 100:9 100:12

3500 G' (Pa)

3250 3000 2750 2500 2250

Tan ()

0

0.19 0.18 0.17 0.16 0.15 0.14 0.13 0.12 0.11 0.10

3600

Time (s)

7200

B

0

10800

100:12 100:9 100:6 100:3 100:0

3600

7200

10800

Time (s) Fig.2 Changes in the storage moduli (G) and tan δ of RS-PRBPH-1 mixtures with mass ratios of 100:0, 100:3, 100:6, 100:9, and 100:12 during an isothermal time sweep step at 4 °C for 3 h.

31

6000 5000

21.62%

Intensity (Counts)

NRS 4000 15.41%

3000

13.88%

100:0

2000

11.57%

100:3 100:6 100:9 100:12

1000 0 0

5

7.92% 4.86%

10

15

20

25

30

35

40

Scattering Angle

Fig.3 XRD patterns of pristine native RS (NRS) and GRS containing various amounts of PRBPH-1 after storage at 4 °C for 14 days.

32

A

B

C

D

Fig.4 CLSM micrographs of RS and PRBPH-1 mixtures with mass ratios of (A) 100:6 and (B) 100:12. Images (C) and (D) are magnified views of the RS-PRBPH-1 treatment with a mass ratio of 100:12 at the focal plane and at 5 µm below the focal plane. Scale bars＝50 µm for A and B.

33

A

B

C

D

Fig.5 AFM topographic images of (A) PRBPH-1 and mixtures of RS and PRBPH-1 with mass ratios of (B) 100:0, (C) 100:6, and (D) 100:12.

34

A1

A2

B1

B2

C1

C2

a1

b2 b1

c1 c2

Fig.6 Scanning electron micrographs of RS and RS-PRBPH-1 mixtures with mass ratios of 100:0 (A1, 500×; A2, 1000×), 100:6 (B1, 500×; B2, 1000×), and 100:12 (C1, 500×; C2, 1000×).

35

Table 1. Gelatinization temperature and enthalpy of RS-PRBPH-1 mixtures with various ratios Mass ratio of RS:PRBPH-1 100:0 100:3 100:6 100:9 100:12

To (°C)*

Tp (°C)* d

64.8±1.06 66.8±1.08c 67.8±0.78bc 68.4±0.51ab 69.4±0.50a

△ H*

Tc (°C)* d

71.1±0.68 72.6±0.75cd 73.8±1.15bc 74.7±0.51ab 76.2±1.12a

a

82.5±0.95 81.47±1.05a 79.1±0.87b 78.5±0.78b 78.2±0.50b

11.46±1.05a 10.56±0.56a 9.12±0.88b 8.58±0.33bc 7.44±0.78c

*Numbers are the mean ± SD (n = 3). Different superscript letters represent significant differences in the mean within the same column (p < 0.05).

36

Table 2. Change in the retrogradation enthalpy and Avrami recrystallization kinetic parameters of RS-PRBPH-1 mixtures after heating from 25 to 110 °C at 5 °C/min and storage at 4 °C for 1, 3, 5, 7, 14, 21 and 28 days Mass ratio of RS:PRBPH-1 100:0 100:3 100:6 100:9 100:12

Enthalpy changes (J/g)* 1 d* 3d 2.36±0.33a 3.50±0.28a b 1.77±0.11 2.78±0.31b 1.30±0.09c 2.43±0.32b d 0.56±0.09 1.16±0.05c 0.38±0.07d 0.56±0.10d

5d 4.15±0.48a 3.42±0.48b 2.90±0.33b 1.50±0.03c 1.17±0.13c

7d 4.89±0.15a 3.96±0.28b 3.23±0.24c 1.63±0.04d 1.66±0.12d

14 d 5.71±0.10a 4.97±0.21b 3.95±0.21c 2.05±0.09d 1.97±0.03d

21 d 6.48±0.29a 5.81±0.12b 4.59±0.12c 2.39±0.22d 2.15±0.07d

28 d 7.12±0.25a 6.47±0.25b 5.05±0.25c 2.95±0.13d 2.36±0.19e

Avrami parameters n K (h-n) r2 0.576 0.375 0.9861 0.630 0.300 0.9835 0.653 0.293 0.9909 0.654 0.227 0.9919 0.934 0.149 0.9471

*Numbers are the mean ± SD (n = 3). Different superscript letters represent significant differences in the mean within the same column (p < 0.05).

37