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Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch
BIOMAC-13865; No of Pages 7 International Journal of Biological Macromolecules xxx (xxxx) xxx
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch Min Zhang a,b, Chao Sun c, Xiaorui Wang a,b, Naifu Wang a,b,⁎, Yibin Zhou a,b a b c
Anhui Province Engineering Laboratory of Agricultural Products Processing, Anhui Agricultural University, Hefei 230036, China Department of Food Science and Engineering, Anhui Agricultural University, Hefei 230036, China Anhui Wanxue Food Co., Ltd., Huaibei 235000, China
a r t i c l e
i n f o
Article history: Received 7 August 2019 Received in revised form 10 October 2019 Accepted 9 November 2019 Available online xxxx Keywords: Wheat starch Rice protein hydrolysates Retrogradation Rheological properties Thermal properties Water mobility
a b s t r a c t In this study, the effect of rice protein hydrolysate (RPH) on the short-term and long-term retrogradation of wheat starch (WS) was investigated. Among the RPH produced using different proteolytic enzymes (alcalase, papain and protamex), WS incorporated protamex-hydrolyzed rice protein which hydrolyzed for 2 h (PRPH-2) exhibited the lowest gel hardness after storage for 7 and 14 days. Compared with native WS, rapid viscosity analyzer (RVA) analysis showed PRPH-2 addition significantly lowered peak viscosity, breakdown and setback values (P b0.05), whereas it increased pasting temperature of WS. Dynamic viscoelastic measurements indicated the presence of PRPH-2 resulted in a decrease in the storage modulus (G') and an increase in the loss tangent (tan δ). Differential scanning calorimetry (DSC) showed that PRPH-2 significantly decreased the retrogradation enthalpy (P b 0.05), enhanced the Avrami exponent (n), and decreased the crystallization rate constant (k) during 28 days of storage. In addition, low field nuclear magnetic resonance (LF-NMR) results showed that PRPH-2 could promote the mobility of water molecules in WS gel. These findings indicated that PRPH-2 may be used to inhibit the short-term and long-term retrogradation of WS, which can be potently used as a natural additive to improve the quality of wheat products. © 2018 Published by Elsevier B.V.
1. Introduction Wheat flour is the primary material used in the preparation of bread, noodles, cakes, tortillas, and many other kinds of foods. These foods supply the daily calories as an important staple food in most of countries [1]. In wheat flour, starch is the main ingredient which comprises of around 70–75% of flour. Because gelatinized starch is subjected to retrogradation during storage, the pliable, soft and elastic wheat products will become rigid, tough and fragmentation [2]. These changes can severely deteriorate the quality of wheat products and shorten their shelf-life, resulting in the food wastage and economic losses [3,4]. Therefore, it is vital to find an effective way to inhibit the starch retrogradation for improving the quality of wheat products during storage. Anti-retrogradation of starch is a well studied topic and protein hydrolysates have been shown to have this activity. When protein hydrolysates are incorporated into food products, their components may interact with other ingredients, such as starch. Niu et al. [5] reported that the addition of porcine plasma protein hydrolysates could ⁎ Corresponding author at: Anhui Province Engineering Laboratory of Agricultural Products Processing, Anhui Agricultural University, Hefei 230036, China. E-mail address: [email protected] (N. Wang).
obviously inhibit long-term retrogradation of corn starch. Xiao and Zhong [6] found that anti-listerial grass carp protein hydrolysate could significantly influence the short-term and long-term retrogradation of gelatinized rice starch. Niu et al. [2] reported that rice bran protein hydrolysates could inhibit the short-term and long-term retrogradation of gelatinized rice starch. Rice is the staple food for more than 3.5 billion people of the world and plays a critical role in food security for more than half of the world's population [7]. During the production of rice, a large amount of byproducts such as broken rice and rice bran are produced. In the past, broken rice has often been used as fodder for animals, which lowered its economic value. The protein digestibility and biological value of rice have been reported to be higher than those of the other major cereals (i.e., wheat, corn and barley) [8]. Also, rice proteins are generally regarded as hypoallergenic, with several studies suggesting that rice proteins or protein-based hydrolysates have anti-oxidative, antihypertensive, anticancer and anti-obesity activities [9]. If rice protein hydrolysates can retard the retrogradation of starch, it may be served as a natural additive to improve the nutrition and quality of wheat products. Therefore, the objective of this study was to prepare RPH to inhibit the retrogradation of WS using food-grade proteolytic enzymes.
https://doi.org/10.1016/j.ijbiomac.2019.11.084 0141-8130/© 2018 Published by Elsevier B.V.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
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Furthermore, the possible impacting mechanism about RPH to inhibit WS retrogradation was investigated using dynamic rheology, differential scanning calorimetry (DSC), and low field nuclear magnetic resonance (LF-NMR). These results may supply some useful information about the method to retard starch retrogradation and provide a potential application in wheat-based foods such as bread, cakes, noodles, and steamed bread to extend their shelf-life. 2. Materials and methods 2.1. Materials Native wheat starch (WS, amylose content of 29.5%) was purchased from Sigma Co., Ltd. (Shanghai, China). Rice protein (protein content of 71.5%) was obtained from Xian Youshuo Biotechnology Co., Ltd. (Xian, China). Papain (800,000 U/g) and protamex (120,000 U/g) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Alcalase (≥200,000 U/g) was from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals were of analytical grade.
2.5. Rheological properties The rheological properties were performed by a DHR-1 Rheometer (TA Instruments, New Castle, DE, USA) using a parallel-plate geometry with 40 mm of diameter and 1000 μm of gap. The suspensions of WS were prepared by mixing native WS (2.0 g, dry basis) and 0, 2, 4, 6 and 8% (w/w) PRPH-2 at a dry starch weight basis with 20 mL of deionized water for 5 min at ambient conditions using a vortex-5 mixer, followed by heating at 95 °С for 20 min in a water bath. Then, the gelatinized starch sample was immediately transferred to the rheometer plate. After locating the parallel, the superfluous sample was removed. The edge of the sample was painted silicon oil to prevent water evaporation during measurement. Before measurement, sample was cooled to 25 °С and held for 5 min to equilibrate temperature and stress. The storage modulus (G') and the loss tangent (tan δ) were measured by the isothermal time sweep of 5 h at 25 °С during aging. The constant oscillation frequency of 1 Hz and constant oscillation strain of 1%, respectively, which was in the limit of linear viscoelastic region for all samples.
2.2. Preparation of rice protein hydrolysates (RPHs)
2.6. Differential scanning calorimetry (DSC)
RPHs was prepared according to the method of Xiao et al. [10]. First, a suspension of rice protein (50 mg protein/mL) was stirred for 1 h at ambient conditions (25 °С) using a stirrer. Then, the suspension was digested for 0, 1 and 2 h to different degree of hydrolysis using the three commercial proteases (alcalase, papain and protamex) at the optimum pH, temperature conditions and the ratio of enzyme to substrate concentration (E/S) was 1.5/100 (g/g). Meanwhile, the pH of the suspension was adjusted to a constant value with 0.1 mol/L HCl or 0.1 mol/L NaOH during hydrolysis. After hydrolysis, the suspension was heated to 95 °С for 15 min to stop the hydrolysis reaction. Then, the hydrolysate was neutralized to pH 7.0 by adding 0.1 mol/L HCl or 0.1 mol/L NaOH and centrifuged at 4500 rpm for 15 min. The supernatant was lyophilized and sealed to store at 4 °С. The degree of hydrolysis of RPHs was calculated by the method of Klost and Drusch [11].
The retrogradation properties of WS with different ratios of PRPH-2 during storage at 4 °С were determined using a differential scanning calorimeter (DSC8000, Perkin Elmer Instruments Company, Ltd., Shanghai, China). To prepare samples, WS and PRPH-2 mixtures (0, 2, 4, 6 and 8% at dry starch weight basis) of approximately 2 mg were weighed into an aluminum pan, and then 6 μL of deionized water was added to each sample. All of pans were hermetically sealed and allowed to equilibrate for 12 h at 4 °С. An empty pan was used as a reference. The temperature scan was preformed from 25 to 110 °С at a constant heating rate of 10 °С/min. The onset temperature (T0), peak temperature (Tp), end temperature (Tc) and gelatinization enthalpy (ΔH) were calculated. After the first-run heating, all of the above gelatinized samples were stored at 4 °С for 1, 3, 5, 7, 14, 21 and 28 days to perform the retrogradation process [12]. Then, the stored samples were rescanned at the same heating rate over the same temperature range to monitor ΔHt. The Avrami equation [13] (Eq. (1)) was applied by many researchers to evaluate the kinetic of starch retrogradation (especially amylopectin) in order to provide a convenient empirical way of representing the process of starch retrogradation. The model is described as follows:
2.3. Measurement of the anti-retrogradation activity of RPHs The hardness of texture profile analysis (TPA) was used to evaluate the anti-retrogradation activity of RPHs. Native WS (2.0 g, dry basis) was suspended in a beaker with 20 mL of deionized water, and 4% (w/ w) RPH at a dry starch weight basis was then added to the starch slurry and mixed for 5 min using a vortex-5 mixer (Haimen Kylin-Bell Lab Instruments Co., Ltd.), followed by heating at 95 °С for 20 min in a water bath (DF-101S, Jiangsu Kexi Instruments Co., Ltd.) with uninterruptedly agitation and cooling for 1 h at room temperature. After storage at 4 °С for 7 and 14 days, the gelatinized starch samples were compressed twice using the TA XTPlus Texture Analyzer (Stable Micro Systems, Goldalming, UK) equipped with a P/0.5 cylindrical probe (diameter of 1.27 cm) at a speed of 1 mm/s and a force of 10 g. The deformation level was 30% of the original sample height.
XðtÞ ¼
ΔHt −ΔH0 ¼ 1− expð−ktn Þ ΔH∞ −ΔH0
ð1Þ
where X(t) and ΔHt are the volume fraction of crystallized amylopectin and the enthalpy change at 1, 3, 5, 7, 14, and 21 days, respectively; ΔH0 is the enthalpy change of the 0 d, generally regarded as zero; ΔH∞ is the limiting enthalpy change (28 d for all samples); and k is the rate constant, and n is the Avrami exponent.
2.4. Pasting properties
2.7. Spin-spin relaxation (T2) measurements
According to the above results of TPA, the rice protein hydrolyzed by protamex at 2 h (PRPH-2) showed the highest anti-retrogradation activity which was used in the following tests. Pasting properties of WS and PRPH-2 mixtures (0, 2, 4, 6 and 8% at dry starch weight basis) were investigated using a Super 3 Rapid Viscosity Analyzer (RVA, Newport Co. Ltd., Australian). The suspension was prepared by mixing 3.5 g of the WS-PRPH-2 mixtures with 25 mL of deionized water in aluminum RVA sample canisters. Then, the slurry was kept at 50 °C for 1 min, and heated from 50 °С to 95 °С within 4 min, held at 95 °С for 2.5 min, allowed to cool to 50 °С within 4 min, and held at 50 °С for 1.5 min.
The transverse relaxation time (T2) was determined using a LF-NMR analyzer (Agilent DD2 600 Hz, Agilent Co., Ltd., US). The sample pastes which prepared as described in rheological properties analysis were transferred to an 8-mm diameter cylindrical NMR glass tube and sealed with parafilm. The tubes were stored at 4 °С for 1, 3, 5, 7, 14, 21 and 28 days. At the conclusion of the storage time, all samples were equilibrated at ambient conditions for 1 h before measurement. Then, the T2 values of all samples were performed using the Carr-PurcellMeiboom-Gill (CPMG) pulse sequence with a 90°-180° pulse spacing of 0.1 ms, and the number of collected echoes was 3000.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
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2.8. Statistical analysis Date were analyzed using SPSS statistical software version 24.0 (SPSS Inc., Chicago, IL, USA). Three independent experimental trials (replications) were used to calculate the mean and standard deviation (SD). Significant differences among means (P b 0.05) were performed by Duncan's multiple range test. 3. Results and discussion 3.1. Effects of RPHs on the hardness of WS gel during storage time The increased hardness of starch-based products is one of the most pronounced effects due to starch retrogradation and it can be used as an index of starch retrogradation [2,14]. The hardness of WS gel with or without the addition of RPHs after storage at 4 °С for 0, 7 and 14 days is shown in Fig. 1. As shown in Fig. 1, an increase in the hardness of WS gel with or without RPHs was observed upon the increasing of storage time. After 14 days of storage, the hardness of WS gel alone increased significantly from 131 to 514 g (P b 0.05). Compared with native WS, WS gel added with RPHs exhibited a softer texture after 7 and 14 days of storage at each time point, indicating the addition of RPHs could effectively retard the starch retrogradation. The results of the present study were similar to the study of Niu et al. [2], who reported that rice starch blended with rice bran protein hydrolysates showed significantly lower hardness than rice starch alone after storage at 4 °C for 14 days. Among the different proteases used in this study, protamexhydrolyzed rice protein at 2 h (PRPH-2) exhibited the lowest gel hardness after storage at 4 °C for 7 and 14 days. In the study of soy protein hydrolysates, Lian et al. [15] found a polypeptide containing seven amino acids was the key component to inhibit maize starch retrogradation. Therefore, PRPH-2 might have the most content of polypeptides which can inhibit WS retrogradation and was selected for further study. 3.2. Effect of PRPH-2 on short-term retrogradation of WS 3.2.1. Pasting properties The effect of PPPH-2 on the pasting properties of WS determined by RVA analysis is shown in Table 1. Compared to WS alone, addition of PRPH-2 at different concentrations led to a significantly decrease in peak viscosity (P b 0.05). Similar results were reported by Kong et al. [16], they found porcine plasma protein hydrolysates could obviously decrease the peak viscosity of corn starch. Ribotta et al. [17] also reported that enzymatic modified pea protein isolate decreased the peak viscosity of corn and cassava starch. Besides the dilution effect on the starch concentration, this decrease might also attribute to the inhibition effect of PRPH-2 on the swelling and gelatinization of starch granule. Likitwattanasade and Hongsprabhas [18] thought that protein can wrap around the surface of starch granules and suppress the gelatinization of starch. This can also be seen in the increase of pasting temperature with the addition of PRPH-2. The breakdown value mainly reflects the stability of starch granules when heating and shear forces are applied. The breakdown values for WS/PRPH-2 mixtures were significantly lower than the control WS (P b 0.05), and the decrease in breakdown was greater at higher concentration of PRPH-2. The reduction of breakdown values elucidated that the starch granules would be more compact with the addition of PRPH-2 and this could result in the decrease of leached amylose, indicating PRPH-2 would exert a protective effect on the granules during heating. Similar to our results, Ribotta et al. [17] found enzymetreated pea protein reduced the breakdown parameter of proteincassava starch gel. However, Kong et al. [16] reported the breakdown of corn starch significantly increased with increasing concentration of porcine plasma protein hydrolysates. The difference between Kong et al. [16] and our study might attribute to the different protein we
Fig. 1. Effects of rice protein (0 h) and rice protein hydrolysate (1 and 2 h) obtained using different enzymes on the retrogradation of gelatinized wheat starch (WS). A, B and C correspond to the alcalase, papain and protamex treatments, respectively. * native WS was tested and served as the control.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
4 t1:1 t1:2
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Table 1 Effect of different ratios of protamex-hydrolyzed rice protein at 2 h (PRPH-2) on pasting properties of wheat starch (WS).
t1:3 t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10
WS WS + 2% PRPH-2* WS + 4% PRPH-2 WS + 6% PRPH-2 WS + 8% PRPH-2
Peak viscosity (cP)
Minimum viscosity (cP)
Final viscosity (cP)
Breakdown (cP)
Total setback (cP)
Pasting temperature (°C)
5235 ± 91.9a 4826 ± 19.1b 4515 ± 57.2c 4196 ± 62.7d 3873 ± 127.7e
3047 ± 45.8a 2969 ± 20.0a 2946 ± 53.5a 2780 ± 51.6b 2543 ± 103.1c
4680 ± 89.6a 4481 ± 26.7b 4332 ± 53.5c 4031 ± 50.5d 3764 ± 102.5e
2188 ± 51.7a 1857 ± 17.7b 1569 ± 64.5c 1416 ± 70.6d 1329 ± 33.8d
1634 ± 54.8a 1512 ± 9.6b 1386 ± 40.6c 1251 ± 54.5d 1220 ± 15.5d
74.55 ± 0.31d 75.90 ± 0.08c 76.60 ± 0.06b 77.23 ± 0.16ab 77.60 ± 0.25a
Values are given as the means ± SD from triplicate determinations; a–e means in the same column with different letters differ significantly (P b 0.05); WS means wheat starch; * means WS mixed with 2, 4, 6, and 8% PRPH-2 at dry starch weight basis.
used. Marco and Rosell [19] found pea and soybean protein isolates didn't cause significantly change on the breakdown value of rice flour, while a significantly decrease was found after addition of egg albumen and whey protein. Liang and King [20] found the negative charged amino acids (aspartic and glutamic acids) had a similar effect as that of the positive charged amino acids (lysine and arginine) for decreasing cooking stability of the rice starch, but opposite effect was found for arginine. The value of setback indicates the recrystallization degree of starch during the cooling process of starch paste, especially the recrystallization and rearrangement level of amylose molecules [21]. As shown in Table 1, setback values significantly decreased (P b 0.05) with the addition of PRPH-2, and this effect was more pronounced as the PRPH-2 ratio increased. The reduction of setback values probably be due to the electrostatic interactions between the polypeptides and WS molecules [22,23], which could be useful for reducing starch retrogradation in wheat products.
3.3. Thermal properties The gelatinization properties of starch samples with or without PRPH-2 determined by DSC are shown in Fig. 3A and Table 2. As shown in Fig. 3A, there was a large gelatinization endothermic peak appearing at about 70 °C for each sample. The gelatinization temperatures (onset temperature, T0; peak temperature, Tp; and end temperature, Tc) increased while gelatinization enthalpy (ΔH) decreased in the presence of PRPH-2. The increases in DSC gelatinization temperatures after addition of PRPH-2 agreed with observations from RVA analysis. Fig. 3B showed the typical thermograms for starch samples with or without PRPH-2 which were stored at 4 °C for 28 days. As can be seen from this figure, an endothermic peak of retrogradation was observed below 60 °C in each sample. The peak retrogradation temperature was lower than the gelatinization temperature, which meant that the
3.2.2. Rheological properties The starch gel with three-dimensional network can be formed due to the rearrangement of starch molecules during cooling and can be determined by the dynamic viscoelastic properties [24]. The storage modulus (G') and loss tangent (tan δ) as a function of time for 5 h at 25 °C are shown in Fig. 2. G' value reflects the elastic property resulting from the junction zones of the three-dimensional network structure of the amylose and swollen granule composite system. The development of the initial gel network structure during storage is governed by the aggregation of amylose. Therefore, G' is usually selected to evaluate the short-term retrogradation of starch [25]. As shown in Fig. 2A, compared with WS alone, addition of PRPH-2 decreased the values of G', which coincided with the low values of peak viscosity obtained by RVA analysis. With the extension of storage time, the pattern of G' of native WS showed an initial swift rise, coming after a slower increase, indicating that the rapid aggregation of amylose occurred at the early stage and consequently formed a three-dimensional gel network. In contrast, the G' values of the WS/ PRPH-2 mixtures initially increased much slower and then remained almost constant, suggesting that PRPH-2 inhibited the aggregation of amylose. The tan δ denotes the associated energy loss versus the energy stored per deformation cycle. A gel with a low tan δ always exhibits elastic behavior, whereas one with a high tan δ displays viscous behavior [24]. As described in Fig. 2B, With the increase of storage time, the tan δ values of all samples decreased, indicating all the gels became more elastic during storage. At each time point, the tan δ values increased with increasing PRPH-2 ratio. Similar findings reported by Niu et al. [2] when rice starch gels were mixed with rice bran protein hydrolysates, indicating a weaker structure formed where the starch network shifted from an elastic-like nature to a more viscous-like. This result confirmed that the aggregation of amylose or the short-term retrogradation of WS was inhibited by PRPH-2 addition. Fig. 2. Changes in the storage moduli (G') (A) and tan δ (B) of wheat starch (WS) mixed with 0, 2, 4, 6 and 8% protamex-hydrolyzed rice protein at 2 h (PRPH-2) during an isothermal time sweep step at 25 °C for 5 h.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
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Fig. 3. The thermograms of gelatinized starches (A) and retrograded starches (B), which were stored at 4 °C for 28 d. WS, wheat starch. PRPH-2, protamex-hydrolyzed rice protein at 2 h. WS mixed with 2, 4, 6, and 8% PRPH-2 at dry starch weight.
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28 days of storage at 4 °С. The ΔHt of all samples increased strongly with increasing storage time. These results indicated that recrystallization occurred during storage and more energy was required to destroy the crystals, leading to an increase in the ΔHt value. This trend was also found in other cereal starch [6,17,28]. Compared with WS, the ΔHt values decreased with the addition of PRPH-2, and lower ΔHt values were obtained when greater amounts of PRPH-2 were added. The enthalpies of the WS-PRPH-2 mixtures decreased from 4.34 to 3.02 J/g as the concentrations of PRPH-2 increased from 2 to 8% when stored for 28 days. These results suggested that PRPH-2 could inhibit the retrogradation of WS especially the crystallization of amylopectin. Some peptides, such as anti-listerial grass carp protein hydrolysate and porcine plasma protein hydrolysates were also found to have the ability to decrease the retrogradation enthalpy of starch [5,6]. According to previous studies, the retrogradation process of gelatinized starch could be analyzed using the theoretical concepts of the polymer, and the Avrami equation has been widely used to study the kinetic of starch retrogradation [3,25]. Table 3 showed the Avrami exponent (n) and rate constant (k) of Avrami model. Parameter k was related to crystal growth and the crystal nucleation constants, while n represented the nature of crystal nucleation and the crystal dimensions [29]. As shown in Table 3, the determination coefficient values (r2, 0.9587–0.9941) were quite close to 1, indicating the experimental data obtained in this study fit with the Avrami theory well. The n values obtained of all samples were smaller than 1, suggesting that the mode of all nucleation in starch recrystallization followed an instantaneous mechanism (nuclei appeared at once) and the growth of crystallites were 1D [5]. Compared with the WS alone, the k values of WS-PRPH-2 mixtures exhibited a slightly decrease, indicating that the recrystallization rate slower. Meanwhile, the n values of WS-PRPH-2 mixtures were higher than that of WS, and the greater the addition amount of PRPH-2, the higher n values obtained. This phenomenon indicated that the nucleation type of starch was transformed from instantaneous nucleation to rod-like growth of crystal [30]. Similar to our results, Niu et al. [5] also found that the addition of porcine plasma protein hydrolysates obviously inhibited corn starch long-term retrogradation using the Avrami equation. These results indicated that PRPH-2 addition could slow down the recrystallization of WS and PRPH-2 might be used to prolong the shelf-life of foods.
3.4. Relaxation properties formation of less perfect crystallites occurred in the retrograded starch samples [26]. The enthalpy values of the retrograded starch indicate the melting of crystallites which consist of the bonding between adjacent double helices during storage. This endothermic peak corresponds to the melting of retrograded amylopectin rather than that of amylose [27]. Table 3 listed the ΔHt of retrograded WS with different ratios of PRPH-2 during t2:1 t2:2 t2:3
Table 2 Effect of different ratios of protamex-hydrolyzed rice protein at 2 h (PRPH-2) for gelatinization temperatures and enthalpy values of wheat starch (WS).
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12
T0 (°С) WS WS + 2% PRPH-2* WS + 4% PRPH-2 WS + 6% PRPH-2 WS + 8% PRPH-2
Tp (°С) d
ΔH (J/g)
Tc (°С) d
c
74.88 ± 0.32 13.62 ± 0.25a 75.97 ± 0.25bc 11.47 ± 0.11b
67.79 ± 0.04 68.44 ± 0.21c
71.45 ± 0.02 72.31 ± 0.24c
69.02 ± 0.14c
72.70 ± 0.13b 76.27 ± 0.13b
11.28 ± 0.16bc
69.50 ± 0.12b 73.11 ± 0.10a 76.71 ± 0.19a
11.07 ± 0.31bc
69.95 ± 0.12a
10.95 ± 0.12c
73.56 ± 0.21a 77.23 ± 0.21a
Values are given as the means ± SD from triplicate determinations; a-d means in the same column with different letters differ significantly (P b 0.05); * means WS mixed with 2, 4, 6, and 8% PRPH-2 at dry starch weight basis.
Water molecules play an important role in starch retrogradation. The constant spin-spin relaxation time (T2) acquired from the LF-NMR experiment can reflect the degree of water mobility in food systems. In general, a lower T2 indicates a close bond between water and solids in food, while higher T2 indicates more free water [31]. As shown in Fig. 4, the T2 values of all samples decreased with the increase of storage time, suggesting a decrease in the overall water mobility. It is known that retrogradation is used to depict changes that occur in gelatinized starch during storage and represents the realignment process of amylose and amylopectin chains from a disordered amorphous state to an ordered crystalline state. The recrystallization process requires the bound water molecules to move into the crystal layer and thus limits the movement of water molecules, which results in a decrease in water mobility [5,31,32]. The T2 values of WS with different ratios of PRPH-2 were all higher than that of the native WS, and the greater the amount added, the higher T2 values were found. These results showed that the addition of PRPH-2 facilitated the water mobility of WS, inhibited water molecules entered into the crystalline structure of starch. Less amorphous area was transformed into recrystallization area and the recrystallization of amylopectin was inhibited. In summary, addition of PRPH-2 into WS paste could retard bound water migration process during storage time and inhibit long-term retrogradation of WS.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
6 t3:1 t3:2 t3:3
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Table 3 Change in the retrogradation enthalpy and Avrami recrystallization kinetic parameters of retrograded wheat starch (WS) and wheat starch/protamex-hydrolyzed rice protein at 2 h (WS/ PRPH-2) mixtures with different mixing ratios 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.
t3:4
Enthalpy changes (J/g)
t3:5
1d
t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12
WS WS + 2% PRPH-2* WS + 4% PRPH-2 WS + 6% PRPH-2 WS + 8% PRPH-2
Avrami parameters
3d a
2.17 ± 0.15 1.45 ± 0.06b 1.25 ± 0.11b 1.02 ± 0.13bc 0.81 ± 0.05c
5d a
2.99 ± 0.01 1.87 ± 0.10b 1.60 ± 0.14bc 1.56 ± 0.07bc 1.33 ± 0.21c
7d a
3.48 ± 0.21 2.09 ± 0.11b 1.98 ± 0.08b 1.81 ± 0.32b 1.57 ± 0.21b
14d a
3.83 ± 0.13 2.52 ± 0.15b 2.13 ± 0.04bc 2.01 ± 0.09bc 1.89 ± 0.30c
21d a
4.60 ± 0.25 3.03 ± 0.07b 2.76 ± 0.14bc 2.42 ± 0.17c 2.09 ± 0.13c
28d a
5.18 ± 0.12 3.52 ± 0.04b 3.04 ± 0.23b 2.79 ± 0.31bc 2.45 ± 0.09c
a
6.30 ± 0.08 4.34 ± 0.15b 3.79 ± 0.28c 3.45 ± 0.04cd 3.02 ± 0.21d
n
k (h-n)
r2
0.456 0.460 0.470 0.494 0.533
0.401 0.362 0.360 0.344 0.318
0.9905 0.9587 0.9734 0.9941 0.9884
Values are given as the mean ± SD from triplicate determinations; a–d that different letters in the same column differ significantly (P b 0.05). * indicates WS mixed with 2, 4, 6, and 8% PRPH2 at dry starch weight basis.
4. Conclusions In the present study, the RPH was produced using different proteolytic enzymes (alcalase, papain and protamex). Findings by the TPA assay showed that the protamex-hydrolyzed rice protein at 2 h (PRPH-2) possessed the highest anti-retrogradation activity. Adding PRPH-2 to WS reduced peak viscosity, minimum viscosity, final viscosity, breakdown and setback values by RVA analysis. Dynamic time sweep analysis of gelatinized samples at 25 °С for 5 h showed a decrease in the storage modulus (G') and an increase in the loss tangent (tan δ) in the presence of PRPH-2, which meant the short-term retrogradation of WS was inhibited. DSC analysis showed that PRPH-2 significantly decreased the retrogradation enthalpy, enhanced the Avrami exponent (n), and decreased the crystallization rate constant (k) during the 28 days of storage at 4 °С. The results of LF-NMR also suggested that the addition of PRPH-2 could increase the water mobility of WS gel during storage time. In summary, this study demonstrated that the addition of PRPH-2 to WS not only could inhibit the short-term retrogradation of amylose, but also could retard the long-term retrogradation of amylopectin. Acknowledgements This study on the financial support of Anhui Natural Science Foundation (11008761), Anhui Science and Technology Plan Project (1704a07020098) and (NIlj20170144).
Fig. 4. The constant spin–spin relaxation time (T2) of retrograded wheat starch (WS) and wheat starch/protamex-hydrolyzed rice protein at 2 h (WS/PRPH-2) mixtures with different mixing ratios stored at 4 °C for the required time.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch Min Zhang a,b, Chao Sun c, Xiaorui Wang a,b, Naifu Wang a,b,⁎, Yibin Zhou a,b a b c
Anhui Province Engineering Laboratory of Agricultural Products Processing, Anhui Agricultural University, Hefei 230036, China Department of Food Science and Engineering, Anhui Agricultural University, Hefei 230036, China Anhui Wanxue Food Co., Ltd., Huaibei 235000, China
a r t i c l e
i n f o
Article history: Received 7 August 2019 Received in revised form 10 October 2019 Accepted 9 November 2019 Available online xxxx Keywords: Wheat starch Rice protein hydrolysates Retrogradation Rheological properties Thermal properties Water mobility
a b s t r a c t In this study, the effect of rice protein hydrolysate (RPH) on the short-term and long-term retrogradation of wheat starch (WS) was investigated. Among the RPH produced using different proteolytic enzymes (alcalase, papain and protamex), WS incorporated protamex-hydrolyzed rice protein which hydrolyzed for 2 h (PRPH-2) exhibited the lowest gel hardness after storage for 7 and 14 days. Compared with native WS, rapid viscosity analyzer (RVA) analysis showed PRPH-2 addition significantly lowered peak viscosity, breakdown and setback values (P b0.05), whereas it increased pasting temperature of WS. Dynamic viscoelastic measurements indicated the presence of PRPH-2 resulted in a decrease in the storage modulus (G') and an increase in the loss tangent (tan δ). Differential scanning calorimetry (DSC) showed that PRPH-2 significantly decreased the retrogradation enthalpy (P b 0.05), enhanced the Avrami exponent (n), and decreased the crystallization rate constant (k) during 28 days of storage. In addition, low field nuclear magnetic resonance (LF-NMR) results showed that PRPH-2 could promote the mobility of water molecules in WS gel. These findings indicated that PRPH-2 may be used to inhibit the short-term and long-term retrogradation of WS, which can be potently used as a natural additive to improve the quality of wheat products. © 2018 Published by Elsevier B.V.
1. Introduction Wheat flour is the primary material used in the preparation of bread, noodles, cakes, tortillas, and many other kinds of foods. These foods supply the daily calories as an important staple food in most of countries [1]. In wheat flour, starch is the main ingredient which comprises of around 70–75% of flour. Because gelatinized starch is subjected to retrogradation during storage, the pliable, soft and elastic wheat products will become rigid, tough and fragmentation [2]. These changes can severely deteriorate the quality of wheat products and shorten their shelf-life, resulting in the food wastage and economic losses [3,4]. Therefore, it is vital to find an effective way to inhibit the starch retrogradation for improving the quality of wheat products during storage. Anti-retrogradation of starch is a well studied topic and protein hydrolysates have been shown to have this activity. When protein hydrolysates are incorporated into food products, their components may interact with other ingredients, such as starch. Niu et al. [5] reported that the addition of porcine plasma protein hydrolysates could ⁎ Corresponding author at: Anhui Province Engineering Laboratory of Agricultural Products Processing, Anhui Agricultural University, Hefei 230036, China. E-mail address: [email protected] (N. Wang).
obviously inhibit long-term retrogradation of corn starch. Xiao and Zhong [6] found that anti-listerial grass carp protein hydrolysate could significantly influence the short-term and long-term retrogradation of gelatinized rice starch. Niu et al. [2] reported that rice bran protein hydrolysates could inhibit the short-term and long-term retrogradation of gelatinized rice starch. Rice is the staple food for more than 3.5 billion people of the world and plays a critical role in food security for more than half of the world's population [7]. During the production of rice, a large amount of byproducts such as broken rice and rice bran are produced. In the past, broken rice has often been used as fodder for animals, which lowered its economic value. The protein digestibility and biological value of rice have been reported to be higher than those of the other major cereals (i.e., wheat, corn and barley) [8]. Also, rice proteins are generally regarded as hypoallergenic, with several studies suggesting that rice proteins or protein-based hydrolysates have anti-oxidative, antihypertensive, anticancer and anti-obesity activities [9]. If rice protein hydrolysates can retard the retrogradation of starch, it may be served as a natural additive to improve the nutrition and quality of wheat products. Therefore, the objective of this study was to prepare RPH to inhibit the retrogradation of WS using food-grade proteolytic enzymes.
https://doi.org/10.1016/j.ijbiomac.2019.11.084 0141-8130/© 2018 Published by Elsevier B.V.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
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Furthermore, the possible impacting mechanism about RPH to inhibit WS retrogradation was investigated using dynamic rheology, differential scanning calorimetry (DSC), and low field nuclear magnetic resonance (LF-NMR). These results may supply some useful information about the method to retard starch retrogradation and provide a potential application in wheat-based foods such as bread, cakes, noodles, and steamed bread to extend their shelf-life. 2. Materials and methods 2.1. Materials Native wheat starch (WS, amylose content of 29.5%) was purchased from Sigma Co., Ltd. (Shanghai, China). Rice protein (protein content of 71.5%) was obtained from Xian Youshuo Biotechnology Co., Ltd. (Xian, China). Papain (800,000 U/g) and protamex (120,000 U/g) were purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). Alcalase (≥200,000 U/g) was from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals were of analytical grade.
2.5. Rheological properties The rheological properties were performed by a DHR-1 Rheometer (TA Instruments, New Castle, DE, USA) using a parallel-plate geometry with 40 mm of diameter and 1000 μm of gap. The suspensions of WS were prepared by mixing native WS (2.0 g, dry basis) and 0, 2, 4, 6 and 8% (w/w) PRPH-2 at a dry starch weight basis with 20 mL of deionized water for 5 min at ambient conditions using a vortex-5 mixer, followed by heating at 95 °С for 20 min in a water bath. Then, the gelatinized starch sample was immediately transferred to the rheometer plate. After locating the parallel, the superfluous sample was removed. The edge of the sample was painted silicon oil to prevent water evaporation during measurement. Before measurement, sample was cooled to 25 °С and held for 5 min to equilibrate temperature and stress. The storage modulus (G') and the loss tangent (tan δ) were measured by the isothermal time sweep of 5 h at 25 °С during aging. The constant oscillation frequency of 1 Hz and constant oscillation strain of 1%, respectively, which was in the limit of linear viscoelastic region for all samples.
2.2. Preparation of rice protein hydrolysates (RPHs)
2.6. Differential scanning calorimetry (DSC)
RPHs was prepared according to the method of Xiao et al. [10]. First, a suspension of rice protein (50 mg protein/mL) was stirred for 1 h at ambient conditions (25 °С) using a stirrer. Then, the suspension was digested for 0, 1 and 2 h to different degree of hydrolysis using the three commercial proteases (alcalase, papain and protamex) at the optimum pH, temperature conditions and the ratio of enzyme to substrate concentration (E/S) was 1.5/100 (g/g). Meanwhile, the pH of the suspension was adjusted to a constant value with 0.1 mol/L HCl or 0.1 mol/L NaOH during hydrolysis. After hydrolysis, the suspension was heated to 95 °С for 15 min to stop the hydrolysis reaction. Then, the hydrolysate was neutralized to pH 7.0 by adding 0.1 mol/L HCl or 0.1 mol/L NaOH and centrifuged at 4500 rpm for 15 min. The supernatant was lyophilized and sealed to store at 4 °С. The degree of hydrolysis of RPHs was calculated by the method of Klost and Drusch [11].
The retrogradation properties of WS with different ratios of PRPH-2 during storage at 4 °С were determined using a differential scanning calorimeter (DSC8000, Perkin Elmer Instruments Company, Ltd., Shanghai, China). To prepare samples, WS and PRPH-2 mixtures (0, 2, 4, 6 and 8% at dry starch weight basis) of approximately 2 mg were weighed into an aluminum pan, and then 6 μL of deionized water was added to each sample. All of pans were hermetically sealed and allowed to equilibrate for 12 h at 4 °С. An empty pan was used as a reference. The temperature scan was preformed from 25 to 110 °С at a constant heating rate of 10 °С/min. The onset temperature (T0), peak temperature (Tp), end temperature (Tc) and gelatinization enthalpy (ΔH) were calculated. After the first-run heating, all of the above gelatinized samples were stored at 4 °С for 1, 3, 5, 7, 14, 21 and 28 days to perform the retrogradation process [12]. Then, the stored samples were rescanned at the same heating rate over the same temperature range to monitor ΔHt. The Avrami equation [13] (Eq. (1)) was applied by many researchers to evaluate the kinetic of starch retrogradation (especially amylopectin) in order to provide a convenient empirical way of representing the process of starch retrogradation. The model is described as follows:
2.3. Measurement of the anti-retrogradation activity of RPHs The hardness of texture profile analysis (TPA) was used to evaluate the anti-retrogradation activity of RPHs. Native WS (2.0 g, dry basis) was suspended in a beaker with 20 mL of deionized water, and 4% (w/ w) RPH at a dry starch weight basis was then added to the starch slurry and mixed for 5 min using a vortex-5 mixer (Haimen Kylin-Bell Lab Instruments Co., Ltd.), followed by heating at 95 °С for 20 min in a water bath (DF-101S, Jiangsu Kexi Instruments Co., Ltd.) with uninterruptedly agitation and cooling for 1 h at room temperature. After storage at 4 °С for 7 and 14 days, the gelatinized starch samples were compressed twice using the TA XTPlus Texture Analyzer (Stable Micro Systems, Goldalming, UK) equipped with a P/0.5 cylindrical probe (diameter of 1.27 cm) at a speed of 1 mm/s and a force of 10 g. The deformation level was 30% of the original sample height.
XðtÞ ¼
ΔHt −ΔH0 ¼ 1− expð−ktn Þ ΔH∞ −ΔH0
ð1Þ
where X(t) and ΔHt are the volume fraction of crystallized amylopectin and the enthalpy change at 1, 3, 5, 7, 14, and 21 days, respectively; ΔH0 is the enthalpy change of the 0 d, generally regarded as zero; ΔH∞ is the limiting enthalpy change (28 d for all samples); and k is the rate constant, and n is the Avrami exponent.
2.4. Pasting properties
2.7. Spin-spin relaxation (T2) measurements
According to the above results of TPA, the rice protein hydrolyzed by protamex at 2 h (PRPH-2) showed the highest anti-retrogradation activity which was used in the following tests. Pasting properties of WS and PRPH-2 mixtures (0, 2, 4, 6 and 8% at dry starch weight basis) were investigated using a Super 3 Rapid Viscosity Analyzer (RVA, Newport Co. Ltd., Australian). The suspension was prepared by mixing 3.5 g of the WS-PRPH-2 mixtures with 25 mL of deionized water in aluminum RVA sample canisters. Then, the slurry was kept at 50 °C for 1 min, and heated from 50 °С to 95 °С within 4 min, held at 95 °С for 2.5 min, allowed to cool to 50 °С within 4 min, and held at 50 °С for 1.5 min.
The transverse relaxation time (T2) was determined using a LF-NMR analyzer (Agilent DD2 600 Hz, Agilent Co., Ltd., US). The sample pastes which prepared as described in rheological properties analysis were transferred to an 8-mm diameter cylindrical NMR glass tube and sealed with parafilm. The tubes were stored at 4 °С for 1, 3, 5, 7, 14, 21 and 28 days. At the conclusion of the storage time, all samples were equilibrated at ambient conditions for 1 h before measurement. Then, the T2 values of all samples were performed using the Carr-PurcellMeiboom-Gill (CPMG) pulse sequence with a 90°-180° pulse spacing of 0.1 ms, and the number of collected echoes was 3000.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
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2.8. Statistical analysis Date were analyzed using SPSS statistical software version 24.0 (SPSS Inc., Chicago, IL, USA). Three independent experimental trials (replications) were used to calculate the mean and standard deviation (SD). Significant differences among means (P b 0.05) were performed by Duncan's multiple range test. 3. Results and discussion 3.1. Effects of RPHs on the hardness of WS gel during storage time The increased hardness of starch-based products is one of the most pronounced effects due to starch retrogradation and it can be used as an index of starch retrogradation [2,14]. The hardness of WS gel with or without the addition of RPHs after storage at 4 °С for 0, 7 and 14 days is shown in Fig. 1. As shown in Fig. 1, an increase in the hardness of WS gel with or without RPHs was observed upon the increasing of storage time. After 14 days of storage, the hardness of WS gel alone increased significantly from 131 to 514 g (P b 0.05). Compared with native WS, WS gel added with RPHs exhibited a softer texture after 7 and 14 days of storage at each time point, indicating the addition of RPHs could effectively retard the starch retrogradation. The results of the present study were similar to the study of Niu et al. [2], who reported that rice starch blended with rice bran protein hydrolysates showed significantly lower hardness than rice starch alone after storage at 4 °C for 14 days. Among the different proteases used in this study, protamexhydrolyzed rice protein at 2 h (PRPH-2) exhibited the lowest gel hardness after storage at 4 °C for 7 and 14 days. In the study of soy protein hydrolysates, Lian et al. [15] found a polypeptide containing seven amino acids was the key component to inhibit maize starch retrogradation. Therefore, PRPH-2 might have the most content of polypeptides which can inhibit WS retrogradation and was selected for further study. 3.2. Effect of PRPH-2 on short-term retrogradation of WS 3.2.1. Pasting properties The effect of PPPH-2 on the pasting properties of WS determined by RVA analysis is shown in Table 1. Compared to WS alone, addition of PRPH-2 at different concentrations led to a significantly decrease in peak viscosity (P b 0.05). Similar results were reported by Kong et al. [16], they found porcine plasma protein hydrolysates could obviously decrease the peak viscosity of corn starch. Ribotta et al. [17] also reported that enzymatic modified pea protein isolate decreased the peak viscosity of corn and cassava starch. Besides the dilution effect on the starch concentration, this decrease might also attribute to the inhibition effect of PRPH-2 on the swelling and gelatinization of starch granule. Likitwattanasade and Hongsprabhas [18] thought that protein can wrap around the surface of starch granules and suppress the gelatinization of starch. This can also be seen in the increase of pasting temperature with the addition of PRPH-2. The breakdown value mainly reflects the stability of starch granules when heating and shear forces are applied. The breakdown values for WS/PRPH-2 mixtures were significantly lower than the control WS (P b 0.05), and the decrease in breakdown was greater at higher concentration of PRPH-2. The reduction of breakdown values elucidated that the starch granules would be more compact with the addition of PRPH-2 and this could result in the decrease of leached amylose, indicating PRPH-2 would exert a protective effect on the granules during heating. Similar to our results, Ribotta et al. [17] found enzymetreated pea protein reduced the breakdown parameter of proteincassava starch gel. However, Kong et al. [16] reported the breakdown of corn starch significantly increased with increasing concentration of porcine plasma protein hydrolysates. The difference between Kong et al. [16] and our study might attribute to the different protein we
Fig. 1. Effects of rice protein (0 h) and rice protein hydrolysate (1 and 2 h) obtained using different enzymes on the retrogradation of gelatinized wheat starch (WS). A, B and C correspond to the alcalase, papain and protamex treatments, respectively. * native WS was tested and served as the control.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
4 t1:1 t1:2
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Table 1 Effect of different ratios of protamex-hydrolyzed rice protein at 2 h (PRPH-2) on pasting properties of wheat starch (WS).
t1:3 t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10
WS WS + 2% PRPH-2* WS + 4% PRPH-2 WS + 6% PRPH-2 WS + 8% PRPH-2
Peak viscosity (cP)
Minimum viscosity (cP)
Final viscosity (cP)
Breakdown (cP)
Total setback (cP)
Pasting temperature (°C)
5235 ± 91.9a 4826 ± 19.1b 4515 ± 57.2c 4196 ± 62.7d 3873 ± 127.7e
3047 ± 45.8a 2969 ± 20.0a 2946 ± 53.5a 2780 ± 51.6b 2543 ± 103.1c
4680 ± 89.6a 4481 ± 26.7b 4332 ± 53.5c 4031 ± 50.5d 3764 ± 102.5e
2188 ± 51.7a 1857 ± 17.7b 1569 ± 64.5c 1416 ± 70.6d 1329 ± 33.8d
1634 ± 54.8a 1512 ± 9.6b 1386 ± 40.6c 1251 ± 54.5d 1220 ± 15.5d
74.55 ± 0.31d 75.90 ± 0.08c 76.60 ± 0.06b 77.23 ± 0.16ab 77.60 ± 0.25a
Values are given as the means ± SD from triplicate determinations; a–e means in the same column with different letters differ significantly (P b 0.05); WS means wheat starch; * means WS mixed with 2, 4, 6, and 8% PRPH-2 at dry starch weight basis.
used. Marco and Rosell [19] found pea and soybean protein isolates didn't cause significantly change on the breakdown value of rice flour, while a significantly decrease was found after addition of egg albumen and whey protein. Liang and King [20] found the negative charged amino acids (aspartic and glutamic acids) had a similar effect as that of the positive charged amino acids (lysine and arginine) for decreasing cooking stability of the rice starch, but opposite effect was found for arginine. The value of setback indicates the recrystallization degree of starch during the cooling process of starch paste, especially the recrystallization and rearrangement level of amylose molecules [21]. As shown in Table 1, setback values significantly decreased (P b 0.05) with the addition of PRPH-2, and this effect was more pronounced as the PRPH-2 ratio increased. The reduction of setback values probably be due to the electrostatic interactions between the polypeptides and WS molecules [22,23], which could be useful for reducing starch retrogradation in wheat products.
3.3. Thermal properties The gelatinization properties of starch samples with or without PRPH-2 determined by DSC are shown in Fig. 3A and Table 2. As shown in Fig. 3A, there was a large gelatinization endothermic peak appearing at about 70 °C for each sample. The gelatinization temperatures (onset temperature, T0; peak temperature, Tp; and end temperature, Tc) increased while gelatinization enthalpy (ΔH) decreased in the presence of PRPH-2. The increases in DSC gelatinization temperatures after addition of PRPH-2 agreed with observations from RVA analysis. Fig. 3B showed the typical thermograms for starch samples with or without PRPH-2 which were stored at 4 °C for 28 days. As can be seen from this figure, an endothermic peak of retrogradation was observed below 60 °C in each sample. The peak retrogradation temperature was lower than the gelatinization temperature, which meant that the
3.2.2. Rheological properties The starch gel with three-dimensional network can be formed due to the rearrangement of starch molecules during cooling and can be determined by the dynamic viscoelastic properties [24]. The storage modulus (G') and loss tangent (tan δ) as a function of time for 5 h at 25 °C are shown in Fig. 2. G' value reflects the elastic property resulting from the junction zones of the three-dimensional network structure of the amylose and swollen granule composite system. The development of the initial gel network structure during storage is governed by the aggregation of amylose. Therefore, G' is usually selected to evaluate the short-term retrogradation of starch [25]. As shown in Fig. 2A, compared with WS alone, addition of PRPH-2 decreased the values of G', which coincided with the low values of peak viscosity obtained by RVA analysis. With the extension of storage time, the pattern of G' of native WS showed an initial swift rise, coming after a slower increase, indicating that the rapid aggregation of amylose occurred at the early stage and consequently formed a three-dimensional gel network. In contrast, the G' values of the WS/ PRPH-2 mixtures initially increased much slower and then remained almost constant, suggesting that PRPH-2 inhibited the aggregation of amylose. The tan δ denotes the associated energy loss versus the energy stored per deformation cycle. A gel with a low tan δ always exhibits elastic behavior, whereas one with a high tan δ displays viscous behavior [24]. As described in Fig. 2B, With the increase of storage time, the tan δ values of all samples decreased, indicating all the gels became more elastic during storage. At each time point, the tan δ values increased with increasing PRPH-2 ratio. Similar findings reported by Niu et al. [2] when rice starch gels were mixed with rice bran protein hydrolysates, indicating a weaker structure formed where the starch network shifted from an elastic-like nature to a more viscous-like. This result confirmed that the aggregation of amylose or the short-term retrogradation of WS was inhibited by PRPH-2 addition. Fig. 2. Changes in the storage moduli (G') (A) and tan δ (B) of wheat starch (WS) mixed with 0, 2, 4, 6 and 8% protamex-hydrolyzed rice protein at 2 h (PRPH-2) during an isothermal time sweep step at 25 °C for 5 h.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
M. Zhang et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx
Fig. 3. The thermograms of gelatinized starches (A) and retrograded starches (B), which were stored at 4 °C for 28 d. WS, wheat starch. PRPH-2, protamex-hydrolyzed rice protein at 2 h. WS mixed with 2, 4, 6, and 8% PRPH-2 at dry starch weight.
5
28 days of storage at 4 °С. The ΔHt of all samples increased strongly with increasing storage time. These results indicated that recrystallization occurred during storage and more energy was required to destroy the crystals, leading to an increase in the ΔHt value. This trend was also found in other cereal starch [6,17,28]. Compared with WS, the ΔHt values decreased with the addition of PRPH-2, and lower ΔHt values were obtained when greater amounts of PRPH-2 were added. The enthalpies of the WS-PRPH-2 mixtures decreased from 4.34 to 3.02 J/g as the concentrations of PRPH-2 increased from 2 to 8% when stored for 28 days. These results suggested that PRPH-2 could inhibit the retrogradation of WS especially the crystallization of amylopectin. Some peptides, such as anti-listerial grass carp protein hydrolysate and porcine plasma protein hydrolysates were also found to have the ability to decrease the retrogradation enthalpy of starch [5,6]. According to previous studies, the retrogradation process of gelatinized starch could be analyzed using the theoretical concepts of the polymer, and the Avrami equation has been widely used to study the kinetic of starch retrogradation [3,25]. Table 3 showed the Avrami exponent (n) and rate constant (k) of Avrami model. Parameter k was related to crystal growth and the crystal nucleation constants, while n represented the nature of crystal nucleation and the crystal dimensions [29]. As shown in Table 3, the determination coefficient values (r2, 0.9587–0.9941) were quite close to 1, indicating the experimental data obtained in this study fit with the Avrami theory well. The n values obtained of all samples were smaller than 1, suggesting that the mode of all nucleation in starch recrystallization followed an instantaneous mechanism (nuclei appeared at once) and the growth of crystallites were 1D [5]. Compared with the WS alone, the k values of WS-PRPH-2 mixtures exhibited a slightly decrease, indicating that the recrystallization rate slower. Meanwhile, the n values of WS-PRPH-2 mixtures were higher than that of WS, and the greater the addition amount of PRPH-2, the higher n values obtained. This phenomenon indicated that the nucleation type of starch was transformed from instantaneous nucleation to rod-like growth of crystal [30]. Similar to our results, Niu et al. [5] also found that the addition of porcine plasma protein hydrolysates obviously inhibited corn starch long-term retrogradation using the Avrami equation. These results indicated that PRPH-2 addition could slow down the recrystallization of WS and PRPH-2 might be used to prolong the shelf-life of foods.
3.4. Relaxation properties formation of less perfect crystallites occurred in the retrograded starch samples [26]. The enthalpy values of the retrograded starch indicate the melting of crystallites which consist of the bonding between adjacent double helices during storage. This endothermic peak corresponds to the melting of retrograded amylopectin rather than that of amylose [27]. Table 3 listed the ΔHt of retrograded WS with different ratios of PRPH-2 during t2:1 t2:2 t2:3
Table 2 Effect of different ratios of protamex-hydrolyzed rice protein at 2 h (PRPH-2) for gelatinization temperatures and enthalpy values of wheat starch (WS).
t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12
T0 (°С) WS WS + 2% PRPH-2* WS + 4% PRPH-2 WS + 6% PRPH-2 WS + 8% PRPH-2
Tp (°С) d
ΔH (J/g)
Tc (°С) d
c
74.88 ± 0.32 13.62 ± 0.25a 75.97 ± 0.25bc 11.47 ± 0.11b
67.79 ± 0.04 68.44 ± 0.21c
71.45 ± 0.02 72.31 ± 0.24c
69.02 ± 0.14c
72.70 ± 0.13b 76.27 ± 0.13b
11.28 ± 0.16bc
69.50 ± 0.12b 73.11 ± 0.10a 76.71 ± 0.19a
11.07 ± 0.31bc
69.95 ± 0.12a
10.95 ± 0.12c
73.56 ± 0.21a 77.23 ± 0.21a
Values are given as the means ± SD from triplicate determinations; a-d means in the same column with different letters differ significantly (P b 0.05); * means WS mixed with 2, 4, 6, and 8% PRPH-2 at dry starch weight basis.
Water molecules play an important role in starch retrogradation. The constant spin-spin relaxation time (T2) acquired from the LF-NMR experiment can reflect the degree of water mobility in food systems. In general, a lower T2 indicates a close bond between water and solids in food, while higher T2 indicates more free water [31]. As shown in Fig. 4, the T2 values of all samples decreased with the increase of storage time, suggesting a decrease in the overall water mobility. It is known that retrogradation is used to depict changes that occur in gelatinized starch during storage and represents the realignment process of amylose and amylopectin chains from a disordered amorphous state to an ordered crystalline state. The recrystallization process requires the bound water molecules to move into the crystal layer and thus limits the movement of water molecules, which results in a decrease in water mobility [5,31,32]. The T2 values of WS with different ratios of PRPH-2 were all higher than that of the native WS, and the greater the amount added, the higher T2 values were found. These results showed that the addition of PRPH-2 facilitated the water mobility of WS, inhibited water molecules entered into the crystalline structure of starch. Less amorphous area was transformed into recrystallization area and the recrystallization of amylopectin was inhibited. In summary, addition of PRPH-2 into WS paste could retard bound water migration process during storage time and inhibit long-term retrogradation of WS.
Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084
6 t3:1 t3:2 t3:3
M. Zhang et al. / International Journal of Biological Macromolecules xxx (xxxx) xxx
Table 3 Change in the retrogradation enthalpy and Avrami recrystallization kinetic parameters of retrograded wheat starch (WS) and wheat starch/protamex-hydrolyzed rice protein at 2 h (WS/ PRPH-2) mixtures with different mixing ratios 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.
t3:4
Enthalpy changes (J/g)
t3:5
1d
t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12
WS WS + 2% PRPH-2* WS + 4% PRPH-2 WS + 6% PRPH-2 WS + 8% PRPH-2
Avrami parameters
3d a
2.17 ± 0.15 1.45 ± 0.06b 1.25 ± 0.11b 1.02 ± 0.13bc 0.81 ± 0.05c
5d a
2.99 ± 0.01 1.87 ± 0.10b 1.60 ± 0.14bc 1.56 ± 0.07bc 1.33 ± 0.21c
7d a
3.48 ± 0.21 2.09 ± 0.11b 1.98 ± 0.08b 1.81 ± 0.32b 1.57 ± 0.21b
14d a
3.83 ± 0.13 2.52 ± 0.15b 2.13 ± 0.04bc 2.01 ± 0.09bc 1.89 ± 0.30c
21d a
4.60 ± 0.25 3.03 ± 0.07b 2.76 ± 0.14bc 2.42 ± 0.17c 2.09 ± 0.13c
28d a
5.18 ± 0.12 3.52 ± 0.04b 3.04 ± 0.23b 2.79 ± 0.31bc 2.45 ± 0.09c
a
6.30 ± 0.08 4.34 ± 0.15b 3.79 ± 0.28c 3.45 ± 0.04cd 3.02 ± 0.21d
n
k (h-n)
r2
0.456 0.460 0.470 0.494 0.533
0.401 0.362 0.360 0.344 0.318
0.9905 0.9587 0.9734 0.9941 0.9884
Values are given as the mean ± SD from triplicate determinations; a–d that different letters in the same column differ significantly (P b 0.05). * indicates WS mixed with 2, 4, 6, and 8% PRPH2 at dry starch weight basis.
4. Conclusions In the present study, the RPH was produced using different proteolytic enzymes (alcalase, papain and protamex). Findings by the TPA assay showed that the protamex-hydrolyzed rice protein at 2 h (PRPH-2) possessed the highest anti-retrogradation activity. Adding PRPH-2 to WS reduced peak viscosity, minimum viscosity, final viscosity, breakdown and setback values by RVA analysis. Dynamic time sweep analysis of gelatinized samples at 25 °С for 5 h showed a decrease in the storage modulus (G') and an increase in the loss tangent (tan δ) in the presence of PRPH-2, which meant the short-term retrogradation of WS was inhibited. DSC analysis showed that PRPH-2 significantly decreased the retrogradation enthalpy, enhanced the Avrami exponent (n), and decreased the crystallization rate constant (k) during the 28 days of storage at 4 °С. The results of LF-NMR also suggested that the addition of PRPH-2 could increase the water mobility of WS gel during storage time. In summary, this study demonstrated that the addition of PRPH-2 to WS not only could inhibit the short-term retrogradation of amylose, but also could retard the long-term retrogradation of amylopectin. Acknowledgements This study on the financial support of Anhui Natural Science Foundation (11008761), Anhui Science and Technology Plan Project (1704a07020098) and (NIlj20170144).
Fig. 4. The constant spin–spin relaxation time (T2) of retrograded wheat starch (WS) and wheat starch/protamex-hydrolyzed rice protein at 2 h (WS/PRPH-2) mixtures with different mixing ratios stored at 4 °C for the required time.
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Please cite this article as: M. Zhang, C. Sun, X. Wang, et al., Effect of rice protein hydrolysates on the short-term and long-term retrogradation of wheat starch, , https://doi.org/10.1016/j.ijbiomac.2019.11.084