- Email: [email protected]
In vitro digestion of nanoscale starch particles and evolution of thermal, morphological, and structural characteristics
Food Hydrocolloids 61 (2016) 344e350
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
In vitro digestion of nanoscale starch particles and evolution of thermal, morphological, and structural characteristics Chengzhen Liu, Suisui Jiang, Zhongjie Han, Liu Xiong, Qingjie Sun* School of Food Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong Province, 266109, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2015 Received in revised form 10 April 2016 Accepted 30 May 2016 Available online 31 May 2016
In vitro digestions of starch nanocrystals (SNCs), prepared by acid hydrolysis of native starch and starch nanoparticles (SNPs) fabricated by self-assembly of short glucan chains, were investigated for kinetics of enzymatic hydrolysis. Their thermal, morphological, and structural properties during amylolysis were also compared. The kinetics of enzymatic hydrolysis indicated that the hydrolysis rate of SNPs was lowest in that of SNCs, cooked starch, and native starch. Onset, peak, conclusion temperatures, and enthalpy of gelatinization of SNPs and SNCs decreased during digestion. The A-type crystallization of SNCs contributed to their higher rate of hydrolysis than that of SNPs, which was B-type. The relative crystallinity of SNCs during hydrolysis decreased but that of SNPs increased. The results suggest that SNPs exhibit higher resistance to digestion than SNCs. This finding provides information for diverse potential applications of SNCs and SNPs. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Starch nanocrystals Starch nanoparticles Digestion
1. Introduction Starch is a natural, renewable, and biodegradable polymer, produced by many plants (e.g., wheat, maize, cassava, legumes, potatoes, rice) as a source of stored energy. Starch can be digested in the gastrointestinal tract to provide energy for the body, but without processing (such as cooking) native starch is not easily digested in the human body because of its semi-crystalline structure, which generally slows digestion (Ao, Roberto, Nichols, Rose, Sterchi, & Hamaker, 2012). More digestible and higher hydrolysis rate were found for cooked starchy food before eating. However, because rapid consumption of starch can cause diabetes, heart disease, cardiovascular disease, obesity, and even certain cancers (Rizkalla, Bellisle, & Slama, 2002), slowly digestible starch (SDS) (Lehmann & Robin, 2007) and resistant starch (RS) (Sajilata, Singhal, & Kulkarni, 2006) are attracting considerable research interest. Starch digestibility can be reduced by modifying starch by means of temperature-cycled crystallization (Zeng, Chen, Kong, Gao, Aadil, & Yu, 2015), enzymatic modification (Zhao & Lin, 2009), hydrothermal treatment (Li, Ward, & Gao, 2011), chemical modification (Miao, Li, Jiang, Cui, Zhang, & Jin, 2014), and acid
* Corresponding author. School of Food Science and Engineering, Qingdao Agricultural University, 266109, 700 Changcheng Road, Chengyang District, Qingdao, China. E-mail address: [email protected] (Q. Sun). http://dx.doi.org/10.1016/j.foodhyd.2016.05.039 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
modification (Ozturk, Koksel, & Perry, 2011). Starch nanocrystals (SNCs) and starch nanoparticles (SNPs) represent a new type of modified starch that is fabricated using two general approaches: top-down and bottom-up. SNCs were the earliest nanoscale starch prepared by the traditional acid method (Kim, Lee, Kim, Lim, & Lim, 2012), and our previous work has reported a creative method using enzymolysis and recrystallization (Sun, Li, Dai, Ji, & Xiong, 2014a). SNCs can remarkably enhance the properties of various materials and substances at very low content. Nanocrystals are being used in biomedical (e.g., in new bio-based nanomaterials and as vehicles for carrying bioactive substances and nutraceuticals) and biochemical fields (Zou, Zhang, Huang, Chang, Su, & Yu, 2011). SNPs have been developed as an alternative to conventional carrier systems for active components, and as a nanofiller in degradable material (Eltayeb, Bakhshi, Stride, & Edirisinghe, 2013). Nano-polymeric material is designed to encapsulate active components with a potential for controlled enzymecatalyzed release of molecules (Lomova et al., 2015). As a nanofiller, which can impart the mechanical reinforcer and barrier properties of starch films, nano-polymeric material must be easily decomposable by enzymes or microorganisms. To our knowledge, there are as yet no reports on SNCs and SNPs as nanoscale modified starch particles concerning about their digestibility by a-amylase and amyloglucosidase. The objectives of the present study were as follows: (1) to investigate the kinetics of the digestion of SNCs and SNPs in vitro,
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
and (2) to monitor the progression of microstructural changes, thermal behavior, and crystalline order during in vitro digestion. This will provide theoretical basis for a range of potential applications based on the starch digestibility. 2. Materials and methods 2.1. Materials Native maize starch (NMS) (approximately 2% amylose and 98% amylopectin) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Pullulanase (E.C.3.2.1.41, 6000 ASPU/67 g, 1.15 g/ ml) was obtained from Novozymes Investment Co. Ltd. (Beijing, China). (ASPU is defined as the amount of enzyme that liberates 1.0 mg glucose from starch in 1 min at pH 4.4 and 60 C). Porcine pancreatic a-amylase (10 U/mg) and amyloglucosidase (100,000 U/ ml) purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. were diluted into pancreatic a-amylase solution (290 U/mL) and amyloglucosidase solution (500 U/mL). The solution was used to hydrolyze NMS, SNCs, and SNPs. All other chemicals were of analytical grade and were used without further purification. 2.2. Preparation of starch nanocrystals and starch nanoparticles SNCs were prepared following the method of Jivan, Madadlou, and Yarmand (2013), with some modifications. NMS (147 g) was dispersed in 100 ml of 3.16 N sulfuric acid and incubated at 40 C for five days while shaken at 100 r/min. The solution was centrifuged at 12,000 r/min for 20 min. The sediment was washed repeatedly in double distilled water (12,000 r/min, 20 min) until it reached a neutral pH. The preparations were powdered by freeze-drying before being sealed and stored for further processing. SNPs were prepared in the manner described by Sun et al. (2014a) with some modifications. NMS (20 g) was dispersed in 100 ml of disodium hydrogen phosphate and a citric acid buffer solution (pH 5.0) to make a starch concentration of 20% (w/v), which was then cooked in boiling water, with vigorous stirring for 30 min to ensure that the starch was fully gelatinized (Sun et al., 2014a). The temperature of the cooked starch solution was adjusted to 56 C, and pullulanase (30 ASPU/g of dry starch) was added. After an enzymatic hydrolysis period of 8 h, the reaction was stopped by heating at 100 C for 30 min and then centrifuged at 3000 r/min for 15 min to remove the precipitate. The pellucid solutions containing short glucan chains were cooled to room temperature and then retrograded at 4 C for 12 h. Suspensions were centrifuged and washed several times with distilled water until neutrality and then freeze-dried.
345
named SNCE-10, SNCE-20, SNCE-60, SNCE-120, and SNPE-10, SNPE20, SNPE-60, SNPE-120, and SNPE-180, respectively. To calculate the extent of hydrolysis, soluble sugars in the supernatant were analyzed, using the phenol-sulfuric acid method (Kim, Park, & Lim, 2008). To prepare cooked maize starch (CMS), NMS (4.0 g, dry basis) was cooked with phosphate buffer (400 ml, pH 5.2) in a boiling water bath for 30 min, stirring constantly to fully gelatinize the starch. After cooling to 40 C, the CMS was equilibrated at 37 C for 5 min. The NMS (4 g, dry basis) was dispersed in phosphate buffer (400 ml, pH 5.2) and equilibrated at 37 C for 5 min. The kinetics of the enzymatic hydrolysis of the NMS and CMS were determined using the same method as described above. 2.4. In vitro enzyme hydrolysis characteristics When starch or starch-containing foods are digested in vitro with relatively high enzyme concentrations for long time periods, the rate of reaction decreases with time and plot of the concentration of product formed (or quantity of starch digested) against time is logarithmic. This substrate decay process fits the standard first-order equation and has been used to investigate the kinetics of starch digestion. The data of starch hydrolysis at each incubation time were fitted to the following equation described by Ørskov and McDonald (1979) to estimate the starch digestibility characteristics:
DCt ¼ D 1 ekðdÞt where DCt (%) is the proportion of starch digested at time t (h), fraction D is the potential starch digestibility (%) that will digest at a rate of Kd, and Kd is the digestion rate (h1). The Marquardt method of the SAS PROC NLIN procedure was used. 2.5. Differential scanning calorimetry (DSC) The thermal properties of NMS, SNCs, and SNPs (before and after enzyme hydrolysis) were determined using a differential scanning calorimeter (DSC 1, Mettler-Toledo International Inc., Switzerland). The samples (about 4 mg, dry basis) with excess water (1:2) were conditioned in hermetic aluminum pans and heated at 10 C/min from 20 to 110 C (Sun et al., 2014a). The onset (To), peak (Tp), conclusion temperatures (Tc) and enthalpy of gelatinization (DH) of the samples were recorded. Enthalpy was calculated using the drystarch weight. 2.6. Scanning electron microscopy (SEM)
2.3. Kinetics of enzymatic hydrolysis The kinetics of enzymatic hydrolysis was determined as described by Li et al. (2013), with some modifications. The SNCs (4 g, dry basis) and SNPs (4 g, dry basis) were dispersed in phosphate buffer (400 ml, pH 5.2) by ultrasonic treatment (40,000 Hz, 30 s). Suspensions were equilibrated at 37 C for 5 min. Seventy glass balls (10 mm in diameter) and 100 mL of enzyme solution, containing pancreatic a-amylase (80 ml, 290 U/mL) and amyloglucosidase (20 ml, 500 U/mL), were added to the suspensions at 37 C, with continuous shaking (150 r/min) in a constant temperature shaking water bath. At 10 min intervals, 714 ml of absolute ethanol was added to stop the reaction, and the suspensions were then centrifuged at 12,000 r/min for 15 min. The precipitates were washed three times in double distilled water and converted to powder form by freeze-drying. The SNC and SNP residues hydrolyzed by enzyme for 10 min, 20 min, 60 min, and 120 min were
The morphological properties of SNCs and SNPs (before and after enzyme hydrolysis) were obtained using a scanning electron microscope (Jeol JSM-6100, Jeol Ltd., Tokyo, Japan). The samples were suspended in acetone to obtain a 0.1% (w/v) suspension. Samples for the SEM were prepared by placing one drop of the colloidal solution onto standard carbon-coated copper grids and then vacuum freeze-drying them under an electric bulb for 30 min. 2.7. X-ray diffraction pattern Structural variations in the NMS, SNCs, SNPs, SNC residues (SNCE-10, SNCE-20, SNCE-60, and SNCE-120), and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180) were analyzed using an X-ray diffractometer (D8 Advance, Bruker AXS, Karlsruhe, Germany). The samples were equilibrated at ambient
346
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
temperature (23 C) for 24 h. X-ray diffraction was performed using an X-ray diffractometer and copper-cobalt radiation. Intensity data were collected using copper radiation in the 2q range 4 e35 . Relative crystallinity is defined as the ratio of peak area to the total area, expressed as a percentage. 2.8. Statistical analysis All experiments were conducted at least three times, and mean values and standard errors were determined. The data were subjected to statistical analysis using SPSS 17.0 (SPSS Inc., Chicago, USA). The experimental data were analyzed by analysis of variance (ANOVA), using the Origin Pro 7.5 statistics program, and expressed as mean values ± standard deviation. Differences were considered significant at a level of 95% (p < 0.05). 3. Results and discussion 3.1. Kinetics of enzymatic hydrolysis The kinetics of enzymatic hydrolysis of NMS, CMS, SNCs, and SNPs are shown in Fig. 1. All the samples exhibited two-stage kinetics (0e20 min and 20e180 min) as shown in Table 1. The trend in changes in their amylolysis rates was similar, with a fast rate in the first stage and a slow rate in the second stage. It is known that native granular starch is not easily digested in the human body because of its semicrystalline structure, developed from double helices of linear branched chains of cluster-like organized amylopectin molecules. NMS displayed a slowly digestible rate of 48.87% at 30 min and 64.41% at 180 min, respectively. As expected, when native starch is heated in the presence of water, the crystalline regions unfolded (Kawai, Takato, Sasaki, & Kajiwara, 2012) and digestibility rate of CMS increased remarkably, to 91.34% at 30 min and 99.00% at 180 min, respectively. A similar previous study noted that RS decreased after starch gelatinization (Maribel, Luis, Bellorez, Díaz, & Simsek, 2011). To our surprise, although SNCs had Pe the highest relative crystallinity (62.21%, Section 3.4) among the starch samples, their hydrolysis rate was extremely high (75.89% at 30 min and 96.35% at 180 min). Generally, low-rate digestion rate is proportionally correlated with high relative crystallinity. Miao, Jiang, Zhang, Jin, and Mu (2011) reported that there is a parallel change between RS and relative crystallinity, which coincides with the higher degree of perfection of starch crystallites, the lower susceptibility to amylolytic degradation. It might attribute to the disruption of the granular structure caused by mild acid hydrolysis, as well as an increase in the effective surface area for amylase binding and hydrolysis.
Table 1 Kinetics of enzymatic hydrolysis of native maize starch (NMS), cooked maize starch (CMS), starch nanocrystals (SNCs) and starch nanoparticles (SNPs). Sample
NMS CMS SNCs SNPs
Hydrolysis characteristics D1 (%)
K1 (h1)
D2 (%)
K2 (h1)
42.11 80.48 66.68 34.81
2.16 4.60 3.67 1.83
64.41 99.00 96.35 56.32
0.53 0.86 0.82 0.44
Calculated using the exponential curve equation DCt ¼ D$(1ek(d)t), where DCt is the proportion of starch digested at time t and D is the potential starch digestibility. D1 is the potential starch digestibility in first 20 min. K1 is the the digestion rate in the first minutes. D2 is the potential starch digestibility in 20e180 min. K2 is the the digestion rate in 20e180 min.
Compared with microscale NMS, SNCs exhibited nanoscale lamella with a size range of 70e100 nm and high relative crystallinity of 62.21%. The large surface area and the highly hydrophilic nature of the SNCs may have contributed to increased reactions with amylase as compared to the NMS. As a result, the SNCs showed weak enzyme resistance despite their high relative crystallinity. The results are in line with the findings of a previous study, which found that the amount of rapidly digestible NMS increased from 32.4% to 56.1% after mild acid hydrolysis (Miao et al., 2011). In contrast, the SNPs exhibited the lowest hydrolysis rate (39.09% at 30 min and 56.32% at 180 min). The hydrolysis rate was almost half that of SNCs at incubation times of 30 min and 180 min. SNPs with a size range of 100e300 nm and high relative crystallinity (46.32%, see Section 3.4) were prepared from short glucan chains by debranching and recrystallization. The slower hydrolysis rate of the SNPs by comparison with the CMS may be due to the compact structure of SNPs formed during the recrystallization of short glucan chains, which would also contribute to the resistance to hydrolysis. Similarly, Shi, Chen, Yu, & Gao (2013) and Zhang and Jin (2011) reported that waxy maize starch treated by pullulanase debranching and retrogradation at room temperature produced high content of RS. An earlier report suggested that as the amount of short chains increased, it was more difficult for the enzymes to digest the substrate (Zhang, Sofyan, & Hamaker, 2008). Short linear chains released from a waxy starch may be crystallized to produce products with a high degree of crystallinity (Shi, Cui, Birkett, & Thatcher, 2006). A high relative crystallinity for starch products indicates that they are resistance to enzymolysis. However, although SNCs have a higher relative crystallinity than SNPs, the hydrolysis rate of SNCs is higher than that of SNPs, perhaps because SNCs show an increase in lamellar structure and are A-type, and expose more enzyme-susceptible interior regions than SNPs, which are B-type (see Section 3.4). It is well known that the hydrolysis reaction of native cereal starch (A-type) with pores and channels proceeds from the hilum region toward the outside of the granule. The digestion mechanism of B-type starches without pores produces a different hydrolysis pattern because enzyme digestion begins from the starch surface (Zhang, Ao, & Hamaker, 2006). This explains why B-type SNPs are more resistant to digestion as compared to SNCs. 3.2. Differential scanning calorimetry (DSC)
Fig. 1. Kinetics of enzymatic hydrolysis of native maize starch (A), cooked maize starch (B), starch nanocrystals (C) and starch nanoparticles (D).
The thermal behaviors of NMS, SNCs, and SNC residues (SNCE10, SNCE-20, SNCE-60, and SNCE-120), SNPs, and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180) are
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
347
Table 2 Gelatinization of native maize starch (NMS), starch nanocrystals (SNCs), and SNC residues (SNCE-10, SNCE-20, SNCE-60 and SNCE-120). Sample
To/ C
NMS SNCs SNCE-10 SNCE-20 SNCE-60 SNCE-120
65.81 67.07 61.14 57.37 64.32 65.47
Tp/ C ± ± ± ± ± ±
0.08ab 1.50a 0.08c 1.34d 0.58b 0.58ab
71.74 88.22 75.22 67.43 73.76 72.99
± ± ± ± ± ±
0.49bc 4.72a 0.00b 1.17c 0.02b 0.54b
Tc/ C
TcTo/ C
83.23 ± 4.70b 101.10 ± 0.87a 82.10 ± 0.05b 81.09 ± 0.35b 80.15 ± 1.48b 78.16 ± 0.86b
17.42 34.03 20.96 23.72 15.83 12.69
± ± ± ± ± ±
0.23d 0.67a 0.18c 0.78b 0.39e 0.94f
DH/J/g 15.36 ± 0.51a 18.38 ± 0.84a 4.34 ± 0.28b 4.28 ± 0.91b 2.79 ± 0.57b 2.35 ± 0.26b
Within the same columns, values with different superscripts letters (a, b, c, d, e and f) are significantly different (p < 0.05). SNCE-10, SNCE-20, SNCE-60, SNCE-120: SNC hydrolyzed residues at 10 min, 20 min, 60 min, and 120 min, respectively.
Table 3 Gelatinization of starch nanoparticles (SNPs) and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120 and SNPE-180). Sample SNPs SNPE-10 SNPE-20 SNPE-60 SNPE-120 SNPE-180
To/ C 69.25 56.17 59.52 63.84 62.04 65.40
Tp/ C ± ± ± ± ± ±
a
0.68 0.50f 0.72e 0.96c 0.62d 0.50b
90.89 80.29 84.38 84.26 85.03 86.89
Tc/ C ± ± ± ± ± ±
a
1.21 1.39e 1.10d 1.86d 1.53c 1.91b
TcTo/ C a
108.96 ± 1.11 101.36 ± 1.34c 103.09 ± 1.01b 102.98b ± 1.66c 101.63 ± 1.86c 104.08 ± 1.57b
39.71 45.19 43.57 39.14 39.59 30.68
± ± ± ± ± ±
1.63c 1.26a 1.45b 1.62c 1.37c 1.47d
DH/J/g 16.26 11.14 10.65 11.16 12.25 11.63
± ± ± ± ± ±
0.97a 0.56c 0.88c 0.53c 0.24b 1.11bc
Within the same columns, values with different superscripts letters (a, b, c, d, e and f) are significantly different (p < 0.05). SNPE-10, SNPE-20, SNPE-60, SNPE-120: SNP hydrolyzed residues at 10 min, 20 min, 60 min, 120 min, and 180 min, respectively.
shown in Tables 2 and 3, respectively. Compared to NMS, SNCs exhibited a broader gelatinization peak and higher To, Tp, Tc, and DH. The Tp of SNCs increased to 88.22 C from 71.74 C for NMS. As digestion time increased from 0 to 120 min, the DH of SNC residues decreased from 18.38 to 4.34, 4.28, 2.79, and 2.35 J/g, respectively. Compared with NMS, SNPs also exhibited a broader gelatinization peak and higher To, Tp, and Tc, and Tp increased to 90.89 C. As digestion time increased from 0 to 180 min, the DH of SNP residues decreased from 16.26 to 11.14, 10.65, 11.16, 12.25, and 11.63 J/g, respectively. Compared with NMS, the shift to the higher and wider range of gelatinization temperatures and DH of SNCs and SNPs indicated that they underwent structural rearrangement. In the case of SNCs, the rearrangement was due to the formation of nanocrystals generated by the hydrolysis of amorphous regions of NMS granules, resulting in a rigid crystalline structure. Kim, Park, Kim, and Lim (2013) reported similar results, showing increased temperatures and ranges for gelatinization of SNCs (prepared by cold acid hydrolysis and ultrasonication). When hydrolyzed by enzymes, the DH of SNC residues decreased with the digestion time increasing, indicating that crystal regions of SNCs could be much more easily digested than amorphous regions, and the damage to structure made SNCs easier to gelatinize. Similarly, O’Brien and Wang (2008) showed that waxy and common corn starches exhibited decreased gelatinization enthalpies upon amyloglucosidase hydrolysis. Man et al. (2013a) also reported that rice starch residues exhibited decreased gelatinization enthalpies after 4 h of amyloglucosidase hydrolysis, suggesting that some of the double helices present in both crystalline and non-crystalline regions were destroyed by amyloglucosidase. As for SNPs, the rearrangement was due to the variation of morphology and fluffiness of particles generated by the amylolysis treatment. Gelatinization temperature and DH decreased after hydrolization, which could be attributed to inhomogeneous crystalline organization and ease of water penetration of SNP residues. The decrease in DH of the SNP residues can be explained in terms of decreased ordering and less stable double helical structures through hydrogen bonding and other intermolecular forces (Mutungi, Rost, Onyango, Jaros, & Rohm, 2009).
Compared with SNCs, the gelatinization temperature and broad gelatinization peak of SNPs were slightly higher. However, SNP residues exhibited a broader gelatinization peak and higher Tp, Tc, and DH than those of SNC residues, perhaps because of the higher relative crystallinity (see Section 3.4) of SNP residues, which require more energy to damage residue structure. 3.3. Morphological characterization Figs. 2 and 3 show the SEM images of SNCs and SNPs before and after digestion, respectively. NMS granules exhibit smooth surfaces and globular, polyhedral shapes with diameters of approximately 5e25 mm (Fig. 2). After acid treatment, the NMS granules were degraded to nanocrystals, with small, irregular, and mesh-like structures and a size range of 70e100 nm with larger aggregates. Similar results were reported by Valodkar and Thakore (2011). After enzymatic hydrolysis, the SNC residues were more individualized and monodispersed than those of the SNCs, which were about 80 nm for SNCE-10 and SNCE-20. However, the agglomeration structure of SNC residues was formed after digestion of SNCs for 60 min and 120 min. The decrease in hydrogen bonding between SNC residues may result in the individualization of SNCE-10 and SNCE-20. The agglomeration structure of SNCE-60 and SNCE-120 may be due to the production of smaller SNPs (Fig. 2). Likewise, on (2004) reported that Pohu, Putaux, Planchot, Colonna, & Bule after enzyme hydrolysis, the maltodextrins looked like a fractal network, with “ghosts” of linters sometimes appearing as elongated units. After debranching and retrogradation of gelatinized NMS, spherical SNPs were formed with a size range of 100e300 nm, which was consistent with previous findings (Sun, Gong, Li, & Xiong, 2014b). After enzymatic hydrolysis, the samples were aggregated at a size of 100e500 nm for SNPE-10, SNPE-20, and SNPE-60, which is larger than SNPs. While SNPE-10 and SNPE-20 expressed an irregular and rough surface, SNPE-60 exhibited apparently random holes and a fluffy surface. However, a similar structure of SNPE-120 and SNPE-180, which is smaller than SNPs, formed after digestion for 120 min and 180 min. During amylolysis, the compact structure of SNPs was
348
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
Fig. 2. SEM of native maize starch (NMS), starch nanocrystals (SNCs), and SNC residues (SNCE-10, SNCE-20, SNCE-60, and SNCE-120).
destroyed, resulting in readier swelling of particles, and in consequence, the particle sizes of SNPE-10, SNPE-20, and SNPE-60 increased. 3.4. X-ray diffraction pattern The X-ray diffraction patterns of NMS, SNCs, and SNC residues (SNCE-10, SNCE-20, SNCE-60, and SNCE-120), SNPs, and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180) are presented in Fig. 4. All SNCs and SNC residues samples exhibited an A-type intensity distribution, which is consistent with the characteristic of NMS. Compared to NMC, there was an increase in the peak intensity of SNCs at 15 , 23 (2q), and in the double peak at 16e18 (2q). In contrast, all the SNPs and SNP residue samples
exhibited a B-type X-ray diffraction pattern, with main diffraction peaks at 2q ¼ 17.1, 22.3 , and 23.8 . By comparison with SNPs, the peaks of SNP residues increased slightly. Shi and Gao (2011) also reported that while native waxy rice starch was A-type, debranched short glucan chains recrystallized at 25 C changed to a B-type X-ray pattern. The relative crystallinity of SNCs increased after acid hydrolysis treatment. Jivan et al. (2013) also noted that, in comparison to granular starch, the higher relative crystallinity of particles obtained through acid hydrolysis of starch highlighted the preferential hydrolysis of amorphous regions of starch granules by acid. Xray diffraction peaks of SNC residues at 15 , 23 (2q), and 16e18 (2q) decreased after amylolysis. The relative crystallinity of NMS, SNCs, SNCE-10, SNCE-20, SNCE-60, and SNCE-120 was 36.02, 71.58,
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
349
Fig. 3. SEM of starch nanoparticles (SNPs) and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180).
68.78, 48.34, 28.69, and 10.93%, respectively. This suggested that hydrolysis of the amorphous and semicrystalline regions of SNCs were not the same and that crystalline regions were hydrolyzed faster than amorphous regions. Man et al. (2013b) reported similar results in a study of the morphology and structure of high-amylose rice starch residues hydrolyzed by amyloglucosidase. They observed that both amorphous and semicrystalline regions of starch granules were hydrolyzed by amyloglucosidase but that crystalline regions were hydrolyzed faster than amorphous regions, resulting in gradually decrease in relative crystallinity of starch residues during hydrolysis. However, the relative crystallinity of SNP residues was higher than that of SNPs. The relative crystallinity of SNPs, SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180 was 44.61, 46.32, 45.34, 48.20, 50.99, and 48.89%, respectively. The results suggested that hydrolysis of amorphous and semicrystalline regions of the SNPs differed in that the former was more rapid than the latter. Clearly, then, while amorphous amylose can be
hydrolyzed rapidly, crystalline amylose and complexes are resistant to hydrolysis (Kawai et al., 2012). 4. Conclusions From the present findings, it can be concluded that the hydrolysis rate of SNPs is lower than that of NMS, SNCs, and CMS. Our results suggest that more SNPs remained unhydrolyzed after 120 min than in the case of NMS, SNCs, and CMS. The relative crystallinity of SNPs increased during digestion while the opposite is true for SNCs. To, Tp, and Tc temperatures, and DH of gelatinization of SNPs and SNCs all decreased during digestion. These findings provide useful information, indicating that SNPs with a low hydrolysis rate are suitable for drug and active delivery applications or as nano-resistant starch, and that SNCs with a high hydrolysis rate may be useful as film material for use as a reinforcer.
350
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
Fig. 4. A. X-ray diffraction patterns of native maize starch (a), starch nanocrystals (SNCs, b) and SNC residues at 10 min (c), 20 min (d), 60 min (e), and 120 min (f). B. Xray diffraction patterns of starch nanoparticles (SNPs, a) and SNP residues at 10 min (b), 20 min (c), 60 min (d), and 120 min (e), 180 min (f).
References Ao, Z. H., Roberto, Q. C., Nichols, B. L., Rose, D. R., Sterchi, E. E., & Hamaker, B. R. (2012). The nature of raw starch digestion. Journal of Pediatric Gastroenterology and Nutrition, 55, 42e43. Eltayeb, M., Bakhshi, P. K., Stride, E., & Edirisinghe, M. (2013). Preparation of solid lipid nanoparticles containing active compound by electrohydrodynamic spraying. Food Research International, 53, 88e95. Jivan, M. J., Madadlou, A., & Yarmand, M. (2013). An attempt to cast light into starch nanocrystals preparation and cross-linking. Food Chemistry, 141, 1661e1666. Kawai, K. S., Takato, S., Sasaki, T., & Kajiwara, K. (2012). Complex formation, thermal properties, and in-vitro digestibility of gelatinized potato starchefatty acid mixtures. Food Hydrocolloids, 27, 228e234. Kim, H. Y., Lee, J. H., Kim, J. Y., Lim, W. J., & Lim, S. T. (2012). Characterization of nanoparticles prepared by acid hydrolysis of various starches. Starch/Starke, 64, 367e373. Kim, H. Y., Park, D. J., Kim, J. Y., & Lim, S. T. (2013). Preparation of crystalline starch nanoparticles using cold acid hydrolysis and ultrasonication. Carbohydrate Polymers, 98, 295e301. Kim, J. Y., Park, D. J., & Lim, S. T. (2008). Fragmentation of waxy rice starch granules by enzymatic hydrolysis. Cereal Chemistry, 85, 182e187. Lehmann, U., & Robin, F. (2007). Slowly digestible starch e its structure and health implications: a review. Trends in Food Science & Technology, 18, 346e355. Li, S. L., Ward, R., & Gao, Q. Y. (2011). Effect of heat-moisture treatment on the formation and physicochemical properties of resistant starch from mung bean (Phaseolus radiatus) starch. Food Hydrocolloids, 25, 1702e1709. Li, H. Y., Zhu, Y. Q., Jiao, A. Q., Zhao, J. W., Chen, X. M., Wei, B. X., et al. (2013). Impact
of a-amylase combined with hydrochloric acid hydrolysis on structure and digestion of waxy rice starch. International Journal of Biological Macromolecules, 55, 276e281. Lomova, M. V., Brichkina, A. I., Kiryukhin, M. V., Vasina, E. N., Pavlov, A. M., Gorin, D. A., et al. (2015). Multilayer capsules of bovine serum albumin and tannic acid for controlled release by enzymatic degradation. Applied Material Interfaces, 7, 11732e11740. Man, J. M., Yang, Y., Zhang, H. J., Zhang, C. Q., Wang, F. M., Gu, Y. P., et al. (2013a). Morphology and structural properties of high-amylose rice starch residues hydrolysed by amyloglucosidase. Food Chemistry, 138, 2089e2098. Man, J. M., Yang, Y., Zhang, C. Q., Zhang, F. M., Wang, Y. P., Gu, M. H., et al. (2013b). Morphology and structural characterization of high-amylose rice starch residues hydrolyzed by porcine pancreatic a-amylase. Food Hydrocolloids, 31, 195e203. rez, K. W., Díaz, P. O., & Simsek, S. (2011). Starch Maribel, O. M., Luis, A., Bello-Pe characteristics of bean (Phaseolus vulgaris L.) grown in different localities. Carbohydrate Polymers, 85, 54e64. Miao, M., Jiang, B., Zhang, T., Jin, Z. Y., & Mu, W. M. (2011). Impact of mild acid hydrolysis on structure and digestion properties of waxy maize starch. Food Chemistry, 126, 506e513. Miao, M., Li, R., Jiang, B., Cui, S. W., Zhang, T., & Jin, Z. (2014). Structure and physicochemical properties of octenyl succinic esters of sugary maize soluble starch and waxy maize starch. Food Chemistry, 151, 154e160. Mutungi, C., Rost, F., Onyango, C., Jaros, D., & Rohm, H. (2009). Crystallinity, thermal and morphological characteristics of resistant starch type III produced by hydrothermal treatment of debranched cassava starch. Starch/ €rke, 61, 634e645. Sta Ørskov, E. R., & McDonald, I. (1979). The estimation of protein degradability in the rumen from incubation measurement weighted according to rate of passage. The Journal of Agricultural Science, 92, 499e503. Ozturk, S., Koksel, H., & Perry, K. W. (2011). Production of resistant starch from acidmodified amylotype starches with enhanced functional properties. Journal of Food Engineering, 103, 156e164. O’Brien, S., & Wang, Y. J. (2008). Susceptibility of annealed starches to hydrolysis by amylase and glucoamylase. Carbohydrate Polymers, 72, 597e607. on, A. (2004). Origin of the Pohu, A., Putaux, J. L., Planchot, V., Colonna, P., & Bule limited a-amylolysis of debranched maltodextrins crystallized in the A form: a TEM study on model substrates. Biomacromolecules, 5, 119e125. Rizkalla, S. W., Bellisle, F., & Slama, G. (2002). Health benefits of low glycaemic indexfoods, such as pulses, in diabetic patients and healthy individuals. British Journal of Nutrition, 88, 25e62. Sajilata, M. G., Singhal, R. S., & Kulkarni, P. R. (2006). Resistant starch - a review. Comprehensive Reviews in Food Science and Technology, 5, 1e17. Shi, Y. C., Cui, X. M., Birkett, A. G., & Thatcher, M. (2006). Resistant starch prepared by isoamylase debranching of low amylose starch. US Patent Office, Pat., 7, 081e261. Shi, M. M., Chen, Y., Yu, S. J., & Gao, Q. Y. (2013). Preparation and properties of RS III from waxy maize starch with Pullulanase. Food Hydrocolloids, 33, 19e25. Shi, M. M., & Gao, Q. Y. (2011). Physicochemical properties, structure and in vitro digestion of resistant starch from waxy rice starch. Carbohydrate Polymers, 84(3), 1151e1157. Sun, Q. J., Gong, M., Li, Y., & Xiong, L. (2014b). Effect of retrogradation time on preparation and characterization of proso millet starch nanoparticles. Carbohydrate Polymers, 111, 133e138. Sun, Q. J., Li, G. H., Dai, L., Ji, N., & Xiong, L. (2014a). Green preparation and characterisation of waxy maize starch nanoparticles through enzymolysis and recrystallization. Food Chemitry, 162, 223e228. Valodkar, M., & Thakore, S. (2011). Organically modified nanosized starch derivatives as excellent reinforcing agents for bionanocomposites. Carbohydrate Polymers, 86, 1244e1251. Zeng, F., Chen, F. Q., Kong, F. S., Gao, Q. Y., Aadil, R. M., & Yu, S. J. (2015). Structure and digestibility of debranched and repeatedly crystallized waxy rice starch. Food Chemistry, 187, 348e353. Zhang, G., Ao, Z., & Hamaker, B. R. (2006). Slow digestion property of native cereal starches. Biomacromolecules, 7, 3252e3258. Zhang, H. X., & Jin, Z. Y. (2011). Preparation of resistant starch by hydrolysis of maize starch with pullulanase. Carbohydrate Polymers, 83, 865e867. Zhang, G. Y., Sofyan, M., & Hamaker, B. R. (2008). Slowly digestible state of starch: mechanism of slow digestion property of gelatinized maize starch. Journal of Agricultural and Food Chemistry, 56, 4695e4702. Zhao, X. H., & Lin, Y. (2009). The impact of coupled acid or pullulanase debranching on the formation of resistant starch from maize starch with autoclaving-cooling cycles. European Food Research and Technology, 230, 179e184. Zou, J. W., Zhang, F., Huang, J., Chang, P. R., Su, Z. M., & Yu, J. H. (2011). Effects of starch nanocrystals on structure and properties of waterborne polyurethanebased composites. Carbohydrate Polymers, 85, 824e831.
Contents lists available at ScienceDirect
Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
In vitro digestion of nanoscale starch particles and evolution of thermal, morphological, and structural characteristics Chengzhen Liu, Suisui Jiang, Zhongjie Han, Liu Xiong, Qingjie Sun* School of Food Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong Province, 266109, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 December 2015 Received in revised form 10 April 2016 Accepted 30 May 2016 Available online 31 May 2016
In vitro digestions of starch nanocrystals (SNCs), prepared by acid hydrolysis of native starch and starch nanoparticles (SNPs) fabricated by self-assembly of short glucan chains, were investigated for kinetics of enzymatic hydrolysis. Their thermal, morphological, and structural properties during amylolysis were also compared. The kinetics of enzymatic hydrolysis indicated that the hydrolysis rate of SNPs was lowest in that of SNCs, cooked starch, and native starch. Onset, peak, conclusion temperatures, and enthalpy of gelatinization of SNPs and SNCs decreased during digestion. The A-type crystallization of SNCs contributed to their higher rate of hydrolysis than that of SNPs, which was B-type. The relative crystallinity of SNCs during hydrolysis decreased but that of SNPs increased. The results suggest that SNPs exhibit higher resistance to digestion than SNCs. This finding provides information for diverse potential applications of SNCs and SNPs. © 2016 Elsevier Ltd. All rights reserved.
Keywords: Starch nanocrystals Starch nanoparticles Digestion
1. Introduction Starch is a natural, renewable, and biodegradable polymer, produced by many plants (e.g., wheat, maize, cassava, legumes, potatoes, rice) as a source of stored energy. Starch can be digested in the gastrointestinal tract to provide energy for the body, but without processing (such as cooking) native starch is not easily digested in the human body because of its semi-crystalline structure, which generally slows digestion (Ao, Roberto, Nichols, Rose, Sterchi, & Hamaker, 2012). More digestible and higher hydrolysis rate were found for cooked starchy food before eating. However, because rapid consumption of starch can cause diabetes, heart disease, cardiovascular disease, obesity, and even certain cancers (Rizkalla, Bellisle, & Slama, 2002), slowly digestible starch (SDS) (Lehmann & Robin, 2007) and resistant starch (RS) (Sajilata, Singhal, & Kulkarni, 2006) are attracting considerable research interest. Starch digestibility can be reduced by modifying starch by means of temperature-cycled crystallization (Zeng, Chen, Kong, Gao, Aadil, & Yu, 2015), enzymatic modification (Zhao & Lin, 2009), hydrothermal treatment (Li, Ward, & Gao, 2011), chemical modification (Miao, Li, Jiang, Cui, Zhang, & Jin, 2014), and acid
* Corresponding author. School of Food Science and Engineering, Qingdao Agricultural University, 266109, 700 Changcheng Road, Chengyang District, Qingdao, China. E-mail address: [email protected] (Q. Sun). http://dx.doi.org/10.1016/j.foodhyd.2016.05.039 0268-005X/© 2016 Elsevier Ltd. All rights reserved.
modification (Ozturk, Koksel, & Perry, 2011). Starch nanocrystals (SNCs) and starch nanoparticles (SNPs) represent a new type of modified starch that is fabricated using two general approaches: top-down and bottom-up. SNCs were the earliest nanoscale starch prepared by the traditional acid method (Kim, Lee, Kim, Lim, & Lim, 2012), and our previous work has reported a creative method using enzymolysis and recrystallization (Sun, Li, Dai, Ji, & Xiong, 2014a). SNCs can remarkably enhance the properties of various materials and substances at very low content. Nanocrystals are being used in biomedical (e.g., in new bio-based nanomaterials and as vehicles for carrying bioactive substances and nutraceuticals) and biochemical fields (Zou, Zhang, Huang, Chang, Su, & Yu, 2011). SNPs have been developed as an alternative to conventional carrier systems for active components, and as a nanofiller in degradable material (Eltayeb, Bakhshi, Stride, & Edirisinghe, 2013). Nano-polymeric material is designed to encapsulate active components with a potential for controlled enzymecatalyzed release of molecules (Lomova et al., 2015). As a nanofiller, which can impart the mechanical reinforcer and barrier properties of starch films, nano-polymeric material must be easily decomposable by enzymes or microorganisms. To our knowledge, there are as yet no reports on SNCs and SNPs as nanoscale modified starch particles concerning about their digestibility by a-amylase and amyloglucosidase. The objectives of the present study were as follows: (1) to investigate the kinetics of the digestion of SNCs and SNPs in vitro,
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
and (2) to monitor the progression of microstructural changes, thermal behavior, and crystalline order during in vitro digestion. This will provide theoretical basis for a range of potential applications based on the starch digestibility. 2. Materials and methods 2.1. Materials Native maize starch (NMS) (approximately 2% amylose and 98% amylopectin) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. Pullulanase (E.C.3.2.1.41, 6000 ASPU/67 g, 1.15 g/ ml) was obtained from Novozymes Investment Co. Ltd. (Beijing, China). (ASPU is defined as the amount of enzyme that liberates 1.0 mg glucose from starch in 1 min at pH 4.4 and 60 C). Porcine pancreatic a-amylase (10 U/mg) and amyloglucosidase (100,000 U/ ml) purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. were diluted into pancreatic a-amylase solution (290 U/mL) and amyloglucosidase solution (500 U/mL). The solution was used to hydrolyze NMS, SNCs, and SNPs. All other chemicals were of analytical grade and were used without further purification. 2.2. Preparation of starch nanocrystals and starch nanoparticles SNCs were prepared following the method of Jivan, Madadlou, and Yarmand (2013), with some modifications. NMS (147 g) was dispersed in 100 ml of 3.16 N sulfuric acid and incubated at 40 C for five days while shaken at 100 r/min. The solution was centrifuged at 12,000 r/min for 20 min. The sediment was washed repeatedly in double distilled water (12,000 r/min, 20 min) until it reached a neutral pH. The preparations were powdered by freeze-drying before being sealed and stored for further processing. SNPs were prepared in the manner described by Sun et al. (2014a) with some modifications. NMS (20 g) was dispersed in 100 ml of disodium hydrogen phosphate and a citric acid buffer solution (pH 5.0) to make a starch concentration of 20% (w/v), which was then cooked in boiling water, with vigorous stirring for 30 min to ensure that the starch was fully gelatinized (Sun et al., 2014a). The temperature of the cooked starch solution was adjusted to 56 C, and pullulanase (30 ASPU/g of dry starch) was added. After an enzymatic hydrolysis period of 8 h, the reaction was stopped by heating at 100 C for 30 min and then centrifuged at 3000 r/min for 15 min to remove the precipitate. The pellucid solutions containing short glucan chains were cooled to room temperature and then retrograded at 4 C for 12 h. Suspensions were centrifuged and washed several times with distilled water until neutrality and then freeze-dried.
345
named SNCE-10, SNCE-20, SNCE-60, SNCE-120, and SNPE-10, SNPE20, SNPE-60, SNPE-120, and SNPE-180, respectively. To calculate the extent of hydrolysis, soluble sugars in the supernatant were analyzed, using the phenol-sulfuric acid method (Kim, Park, & Lim, 2008). To prepare cooked maize starch (CMS), NMS (4.0 g, dry basis) was cooked with phosphate buffer (400 ml, pH 5.2) in a boiling water bath for 30 min, stirring constantly to fully gelatinize the starch. After cooling to 40 C, the CMS was equilibrated at 37 C for 5 min. The NMS (4 g, dry basis) was dispersed in phosphate buffer (400 ml, pH 5.2) and equilibrated at 37 C for 5 min. The kinetics of the enzymatic hydrolysis of the NMS and CMS were determined using the same method as described above. 2.4. In vitro enzyme hydrolysis characteristics When starch or starch-containing foods are digested in vitro with relatively high enzyme concentrations for long time periods, the rate of reaction decreases with time and plot of the concentration of product formed (or quantity of starch digested) against time is logarithmic. This substrate decay process fits the standard first-order equation and has been used to investigate the kinetics of starch digestion. The data of starch hydrolysis at each incubation time were fitted to the following equation described by Ørskov and McDonald (1979) to estimate the starch digestibility characteristics:
DCt ¼ D 1 ekðdÞt where DCt (%) is the proportion of starch digested at time t (h), fraction D is the potential starch digestibility (%) that will digest at a rate of Kd, and Kd is the digestion rate (h1). The Marquardt method of the SAS PROC NLIN procedure was used. 2.5. Differential scanning calorimetry (DSC) The thermal properties of NMS, SNCs, and SNPs (before and after enzyme hydrolysis) were determined using a differential scanning calorimeter (DSC 1, Mettler-Toledo International Inc., Switzerland). The samples (about 4 mg, dry basis) with excess water (1:2) were conditioned in hermetic aluminum pans and heated at 10 C/min from 20 to 110 C (Sun et al., 2014a). The onset (To), peak (Tp), conclusion temperatures (Tc) and enthalpy of gelatinization (DH) of the samples were recorded. Enthalpy was calculated using the drystarch weight. 2.6. Scanning electron microscopy (SEM)
2.3. Kinetics of enzymatic hydrolysis The kinetics of enzymatic hydrolysis was determined as described by Li et al. (2013), with some modifications. The SNCs (4 g, dry basis) and SNPs (4 g, dry basis) were dispersed in phosphate buffer (400 ml, pH 5.2) by ultrasonic treatment (40,000 Hz, 30 s). Suspensions were equilibrated at 37 C for 5 min. Seventy glass balls (10 mm in diameter) and 100 mL of enzyme solution, containing pancreatic a-amylase (80 ml, 290 U/mL) and amyloglucosidase (20 ml, 500 U/mL), were added to the suspensions at 37 C, with continuous shaking (150 r/min) in a constant temperature shaking water bath. At 10 min intervals, 714 ml of absolute ethanol was added to stop the reaction, and the suspensions were then centrifuged at 12,000 r/min for 15 min. The precipitates were washed three times in double distilled water and converted to powder form by freeze-drying. The SNC and SNP residues hydrolyzed by enzyme for 10 min, 20 min, 60 min, and 120 min were
The morphological properties of SNCs and SNPs (before and after enzyme hydrolysis) were obtained using a scanning electron microscope (Jeol JSM-6100, Jeol Ltd., Tokyo, Japan). The samples were suspended in acetone to obtain a 0.1% (w/v) suspension. Samples for the SEM were prepared by placing one drop of the colloidal solution onto standard carbon-coated copper grids and then vacuum freeze-drying them under an electric bulb for 30 min. 2.7. X-ray diffraction pattern Structural variations in the NMS, SNCs, SNPs, SNC residues (SNCE-10, SNCE-20, SNCE-60, and SNCE-120), and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180) were analyzed using an X-ray diffractometer (D8 Advance, Bruker AXS, Karlsruhe, Germany). The samples were equilibrated at ambient
346
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
temperature (23 C) for 24 h. X-ray diffraction was performed using an X-ray diffractometer and copper-cobalt radiation. Intensity data were collected using copper radiation in the 2q range 4 e35 . Relative crystallinity is defined as the ratio of peak area to the total area, expressed as a percentage. 2.8. Statistical analysis All experiments were conducted at least three times, and mean values and standard errors were determined. The data were subjected to statistical analysis using SPSS 17.0 (SPSS Inc., Chicago, USA). The experimental data were analyzed by analysis of variance (ANOVA), using the Origin Pro 7.5 statistics program, and expressed as mean values ± standard deviation. Differences were considered significant at a level of 95% (p < 0.05). 3. Results and discussion 3.1. Kinetics of enzymatic hydrolysis The kinetics of enzymatic hydrolysis of NMS, CMS, SNCs, and SNPs are shown in Fig. 1. All the samples exhibited two-stage kinetics (0e20 min and 20e180 min) as shown in Table 1. The trend in changes in their amylolysis rates was similar, with a fast rate in the first stage and a slow rate in the second stage. It is known that native granular starch is not easily digested in the human body because of its semicrystalline structure, developed from double helices of linear branched chains of cluster-like organized amylopectin molecules. NMS displayed a slowly digestible rate of 48.87% at 30 min and 64.41% at 180 min, respectively. As expected, when native starch is heated in the presence of water, the crystalline regions unfolded (Kawai, Takato, Sasaki, & Kajiwara, 2012) and digestibility rate of CMS increased remarkably, to 91.34% at 30 min and 99.00% at 180 min, respectively. A similar previous study noted that RS decreased after starch gelatinization (Maribel, Luis, Bellorez, Díaz, & Simsek, 2011). To our surprise, although SNCs had Pe the highest relative crystallinity (62.21%, Section 3.4) among the starch samples, their hydrolysis rate was extremely high (75.89% at 30 min and 96.35% at 180 min). Generally, low-rate digestion rate is proportionally correlated with high relative crystallinity. Miao, Jiang, Zhang, Jin, and Mu (2011) reported that there is a parallel change between RS and relative crystallinity, which coincides with the higher degree of perfection of starch crystallites, the lower susceptibility to amylolytic degradation. It might attribute to the disruption of the granular structure caused by mild acid hydrolysis, as well as an increase in the effective surface area for amylase binding and hydrolysis.
Table 1 Kinetics of enzymatic hydrolysis of native maize starch (NMS), cooked maize starch (CMS), starch nanocrystals (SNCs) and starch nanoparticles (SNPs). Sample
NMS CMS SNCs SNPs
Hydrolysis characteristics D1 (%)
K1 (h1)
D2 (%)
K2 (h1)
42.11 80.48 66.68 34.81
2.16 4.60 3.67 1.83
64.41 99.00 96.35 56.32
0.53 0.86 0.82 0.44
Calculated using the exponential curve equation DCt ¼ D$(1ek(d)t), where DCt is the proportion of starch digested at time t and D is the potential starch digestibility. D1 is the potential starch digestibility in first 20 min. K1 is the the digestion rate in the first minutes. D2 is the potential starch digestibility in 20e180 min. K2 is the the digestion rate in 20e180 min.
Compared with microscale NMS, SNCs exhibited nanoscale lamella with a size range of 70e100 nm and high relative crystallinity of 62.21%. The large surface area and the highly hydrophilic nature of the SNCs may have contributed to increased reactions with amylase as compared to the NMS. As a result, the SNCs showed weak enzyme resistance despite their high relative crystallinity. The results are in line with the findings of a previous study, which found that the amount of rapidly digestible NMS increased from 32.4% to 56.1% after mild acid hydrolysis (Miao et al., 2011). In contrast, the SNPs exhibited the lowest hydrolysis rate (39.09% at 30 min and 56.32% at 180 min). The hydrolysis rate was almost half that of SNCs at incubation times of 30 min and 180 min. SNPs with a size range of 100e300 nm and high relative crystallinity (46.32%, see Section 3.4) were prepared from short glucan chains by debranching and recrystallization. The slower hydrolysis rate of the SNPs by comparison with the CMS may be due to the compact structure of SNPs formed during the recrystallization of short glucan chains, which would also contribute to the resistance to hydrolysis. Similarly, Shi, Chen, Yu, & Gao (2013) and Zhang and Jin (2011) reported that waxy maize starch treated by pullulanase debranching and retrogradation at room temperature produced high content of RS. An earlier report suggested that as the amount of short chains increased, it was more difficult for the enzymes to digest the substrate (Zhang, Sofyan, & Hamaker, 2008). Short linear chains released from a waxy starch may be crystallized to produce products with a high degree of crystallinity (Shi, Cui, Birkett, & Thatcher, 2006). A high relative crystallinity for starch products indicates that they are resistance to enzymolysis. However, although SNCs have a higher relative crystallinity than SNPs, the hydrolysis rate of SNCs is higher than that of SNPs, perhaps because SNCs show an increase in lamellar structure and are A-type, and expose more enzyme-susceptible interior regions than SNPs, which are B-type (see Section 3.4). It is well known that the hydrolysis reaction of native cereal starch (A-type) with pores and channels proceeds from the hilum region toward the outside of the granule. The digestion mechanism of B-type starches without pores produces a different hydrolysis pattern because enzyme digestion begins from the starch surface (Zhang, Ao, & Hamaker, 2006). This explains why B-type SNPs are more resistant to digestion as compared to SNCs. 3.2. Differential scanning calorimetry (DSC)
Fig. 1. Kinetics of enzymatic hydrolysis of native maize starch (A), cooked maize starch (B), starch nanocrystals (C) and starch nanoparticles (D).
The thermal behaviors of NMS, SNCs, and SNC residues (SNCE10, SNCE-20, SNCE-60, and SNCE-120), SNPs, and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180) are
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
347
Table 2 Gelatinization of native maize starch (NMS), starch nanocrystals (SNCs), and SNC residues (SNCE-10, SNCE-20, SNCE-60 and SNCE-120). Sample
To/ C
NMS SNCs SNCE-10 SNCE-20 SNCE-60 SNCE-120
65.81 67.07 61.14 57.37 64.32 65.47
Tp/ C ± ± ± ± ± ±
0.08ab 1.50a 0.08c 1.34d 0.58b 0.58ab
71.74 88.22 75.22 67.43 73.76 72.99
± ± ± ± ± ±
0.49bc 4.72a 0.00b 1.17c 0.02b 0.54b
Tc/ C
TcTo/ C
83.23 ± 4.70b 101.10 ± 0.87a 82.10 ± 0.05b 81.09 ± 0.35b 80.15 ± 1.48b 78.16 ± 0.86b
17.42 34.03 20.96 23.72 15.83 12.69
± ± ± ± ± ±
0.23d 0.67a 0.18c 0.78b 0.39e 0.94f
DH/J/g 15.36 ± 0.51a 18.38 ± 0.84a 4.34 ± 0.28b 4.28 ± 0.91b 2.79 ± 0.57b 2.35 ± 0.26b
Within the same columns, values with different superscripts letters (a, b, c, d, e and f) are significantly different (p < 0.05). SNCE-10, SNCE-20, SNCE-60, SNCE-120: SNC hydrolyzed residues at 10 min, 20 min, 60 min, and 120 min, respectively.
Table 3 Gelatinization of starch nanoparticles (SNPs) and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120 and SNPE-180). Sample SNPs SNPE-10 SNPE-20 SNPE-60 SNPE-120 SNPE-180
To/ C 69.25 56.17 59.52 63.84 62.04 65.40
Tp/ C ± ± ± ± ± ±
a
0.68 0.50f 0.72e 0.96c 0.62d 0.50b
90.89 80.29 84.38 84.26 85.03 86.89
Tc/ C ± ± ± ± ± ±
a
1.21 1.39e 1.10d 1.86d 1.53c 1.91b
TcTo/ C a
108.96 ± 1.11 101.36 ± 1.34c 103.09 ± 1.01b 102.98b ± 1.66c 101.63 ± 1.86c 104.08 ± 1.57b
39.71 45.19 43.57 39.14 39.59 30.68
± ± ± ± ± ±
1.63c 1.26a 1.45b 1.62c 1.37c 1.47d
DH/J/g 16.26 11.14 10.65 11.16 12.25 11.63
± ± ± ± ± ±
0.97a 0.56c 0.88c 0.53c 0.24b 1.11bc
Within the same columns, values with different superscripts letters (a, b, c, d, e and f) are significantly different (p < 0.05). SNPE-10, SNPE-20, SNPE-60, SNPE-120: SNP hydrolyzed residues at 10 min, 20 min, 60 min, 120 min, and 180 min, respectively.
shown in Tables 2 and 3, respectively. Compared to NMS, SNCs exhibited a broader gelatinization peak and higher To, Tp, Tc, and DH. The Tp of SNCs increased to 88.22 C from 71.74 C for NMS. As digestion time increased from 0 to 120 min, the DH of SNC residues decreased from 18.38 to 4.34, 4.28, 2.79, and 2.35 J/g, respectively. Compared with NMS, SNPs also exhibited a broader gelatinization peak and higher To, Tp, and Tc, and Tp increased to 90.89 C. As digestion time increased from 0 to 180 min, the DH of SNP residues decreased from 16.26 to 11.14, 10.65, 11.16, 12.25, and 11.63 J/g, respectively. Compared with NMS, the shift to the higher and wider range of gelatinization temperatures and DH of SNCs and SNPs indicated that they underwent structural rearrangement. In the case of SNCs, the rearrangement was due to the formation of nanocrystals generated by the hydrolysis of amorphous regions of NMS granules, resulting in a rigid crystalline structure. Kim, Park, Kim, and Lim (2013) reported similar results, showing increased temperatures and ranges for gelatinization of SNCs (prepared by cold acid hydrolysis and ultrasonication). When hydrolyzed by enzymes, the DH of SNC residues decreased with the digestion time increasing, indicating that crystal regions of SNCs could be much more easily digested than amorphous regions, and the damage to structure made SNCs easier to gelatinize. Similarly, O’Brien and Wang (2008) showed that waxy and common corn starches exhibited decreased gelatinization enthalpies upon amyloglucosidase hydrolysis. Man et al. (2013a) also reported that rice starch residues exhibited decreased gelatinization enthalpies after 4 h of amyloglucosidase hydrolysis, suggesting that some of the double helices present in both crystalline and non-crystalline regions were destroyed by amyloglucosidase. As for SNPs, the rearrangement was due to the variation of morphology and fluffiness of particles generated by the amylolysis treatment. Gelatinization temperature and DH decreased after hydrolization, which could be attributed to inhomogeneous crystalline organization and ease of water penetration of SNP residues. The decrease in DH of the SNP residues can be explained in terms of decreased ordering and less stable double helical structures through hydrogen bonding and other intermolecular forces (Mutungi, Rost, Onyango, Jaros, & Rohm, 2009).
Compared with SNCs, the gelatinization temperature and broad gelatinization peak of SNPs were slightly higher. However, SNP residues exhibited a broader gelatinization peak and higher Tp, Tc, and DH than those of SNC residues, perhaps because of the higher relative crystallinity (see Section 3.4) of SNP residues, which require more energy to damage residue structure. 3.3. Morphological characterization Figs. 2 and 3 show the SEM images of SNCs and SNPs before and after digestion, respectively. NMS granules exhibit smooth surfaces and globular, polyhedral shapes with diameters of approximately 5e25 mm (Fig. 2). After acid treatment, the NMS granules were degraded to nanocrystals, with small, irregular, and mesh-like structures and a size range of 70e100 nm with larger aggregates. Similar results were reported by Valodkar and Thakore (2011). After enzymatic hydrolysis, the SNC residues were more individualized and monodispersed than those of the SNCs, which were about 80 nm for SNCE-10 and SNCE-20. However, the agglomeration structure of SNC residues was formed after digestion of SNCs for 60 min and 120 min. The decrease in hydrogen bonding between SNC residues may result in the individualization of SNCE-10 and SNCE-20. The agglomeration structure of SNCE-60 and SNCE-120 may be due to the production of smaller SNPs (Fig. 2). Likewise, on (2004) reported that Pohu, Putaux, Planchot, Colonna, & Bule after enzyme hydrolysis, the maltodextrins looked like a fractal network, with “ghosts” of linters sometimes appearing as elongated units. After debranching and retrogradation of gelatinized NMS, spherical SNPs were formed with a size range of 100e300 nm, which was consistent with previous findings (Sun, Gong, Li, & Xiong, 2014b). After enzymatic hydrolysis, the samples were aggregated at a size of 100e500 nm for SNPE-10, SNPE-20, and SNPE-60, which is larger than SNPs. While SNPE-10 and SNPE-20 expressed an irregular and rough surface, SNPE-60 exhibited apparently random holes and a fluffy surface. However, a similar structure of SNPE-120 and SNPE-180, which is smaller than SNPs, formed after digestion for 120 min and 180 min. During amylolysis, the compact structure of SNPs was
348
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
Fig. 2. SEM of native maize starch (NMS), starch nanocrystals (SNCs), and SNC residues (SNCE-10, SNCE-20, SNCE-60, and SNCE-120).
destroyed, resulting in readier swelling of particles, and in consequence, the particle sizes of SNPE-10, SNPE-20, and SNPE-60 increased. 3.4. X-ray diffraction pattern The X-ray diffraction patterns of NMS, SNCs, and SNC residues (SNCE-10, SNCE-20, SNCE-60, and SNCE-120), SNPs, and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180) are presented in Fig. 4. All SNCs and SNC residues samples exhibited an A-type intensity distribution, which is consistent with the characteristic of NMS. Compared to NMC, there was an increase in the peak intensity of SNCs at 15 , 23 (2q), and in the double peak at 16e18 (2q). In contrast, all the SNPs and SNP residue samples
exhibited a B-type X-ray diffraction pattern, with main diffraction peaks at 2q ¼ 17.1, 22.3 , and 23.8 . By comparison with SNPs, the peaks of SNP residues increased slightly. Shi and Gao (2011) also reported that while native waxy rice starch was A-type, debranched short glucan chains recrystallized at 25 C changed to a B-type X-ray pattern. The relative crystallinity of SNCs increased after acid hydrolysis treatment. Jivan et al. (2013) also noted that, in comparison to granular starch, the higher relative crystallinity of particles obtained through acid hydrolysis of starch highlighted the preferential hydrolysis of amorphous regions of starch granules by acid. Xray diffraction peaks of SNC residues at 15 , 23 (2q), and 16e18 (2q) decreased after amylolysis. The relative crystallinity of NMS, SNCs, SNCE-10, SNCE-20, SNCE-60, and SNCE-120 was 36.02, 71.58,
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
349
Fig. 3. SEM of starch nanoparticles (SNPs) and SNP residues (SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180).
68.78, 48.34, 28.69, and 10.93%, respectively. This suggested that hydrolysis of the amorphous and semicrystalline regions of SNCs were not the same and that crystalline regions were hydrolyzed faster than amorphous regions. Man et al. (2013b) reported similar results in a study of the morphology and structure of high-amylose rice starch residues hydrolyzed by amyloglucosidase. They observed that both amorphous and semicrystalline regions of starch granules were hydrolyzed by amyloglucosidase but that crystalline regions were hydrolyzed faster than amorphous regions, resulting in gradually decrease in relative crystallinity of starch residues during hydrolysis. However, the relative crystallinity of SNP residues was higher than that of SNPs. The relative crystallinity of SNPs, SNPE-10, SNPE-20, SNPE-60, SNPE-120, and SNPE-180 was 44.61, 46.32, 45.34, 48.20, 50.99, and 48.89%, respectively. The results suggested that hydrolysis of amorphous and semicrystalline regions of the SNPs differed in that the former was more rapid than the latter. Clearly, then, while amorphous amylose can be
hydrolyzed rapidly, crystalline amylose and complexes are resistant to hydrolysis (Kawai et al., 2012). 4. Conclusions From the present findings, it can be concluded that the hydrolysis rate of SNPs is lower than that of NMS, SNCs, and CMS. Our results suggest that more SNPs remained unhydrolyzed after 120 min than in the case of NMS, SNCs, and CMS. The relative crystallinity of SNPs increased during digestion while the opposite is true for SNCs. To, Tp, and Tc temperatures, and DH of gelatinization of SNPs and SNCs all decreased during digestion. These findings provide useful information, indicating that SNPs with a low hydrolysis rate are suitable for drug and active delivery applications or as nano-resistant starch, and that SNCs with a high hydrolysis rate may be useful as film material for use as a reinforcer.
350
C. Liu et al. / Food Hydrocolloids 61 (2016) 344e350
Fig. 4. A. X-ray diffraction patterns of native maize starch (a), starch nanocrystals (SNCs, b) and SNC residues at 10 min (c), 20 min (d), 60 min (e), and 120 min (f). B. Xray diffraction patterns of starch nanoparticles (SNPs, a) and SNP residues at 10 min (b), 20 min (c), 60 min (d), and 120 min (e), 180 min (f).
References Ao, Z. H., Roberto, Q. C., Nichols, B. L., Rose, D. R., Sterchi, E. E., & Hamaker, B. R. (2012). The nature of raw starch digestion. Journal of Pediatric Gastroenterology and Nutrition, 55, 42e43. Eltayeb, M., Bakhshi, P. K., Stride, E., & Edirisinghe, M. (2013). Preparation of solid lipid nanoparticles containing active compound by electrohydrodynamic spraying. Food Research International, 53, 88e95. Jivan, M. J., Madadlou, A., & Yarmand, M. (2013). An attempt to cast light into starch nanocrystals preparation and cross-linking. Food Chemistry, 141, 1661e1666. Kawai, K. S., Takato, S., Sasaki, T., & Kajiwara, K. (2012). Complex formation, thermal properties, and in-vitro digestibility of gelatinized potato starchefatty acid mixtures. Food Hydrocolloids, 27, 228e234. Kim, H. Y., Lee, J. H., Kim, J. Y., Lim, W. J., & Lim, S. T. (2012). Characterization of nanoparticles prepared by acid hydrolysis of various starches. Starch/Starke, 64, 367e373. Kim, H. Y., Park, D. J., Kim, J. Y., & Lim, S. T. (2013). Preparation of crystalline starch nanoparticles using cold acid hydrolysis and ultrasonication. Carbohydrate Polymers, 98, 295e301. Kim, J. Y., Park, D. J., & Lim, S. T. (2008). Fragmentation of waxy rice starch granules by enzymatic hydrolysis. Cereal Chemistry, 85, 182e187. Lehmann, U., & Robin, F. (2007). Slowly digestible starch e its structure and health implications: a review. Trends in Food Science & Technology, 18, 346e355. Li, S. L., Ward, R., & Gao, Q. Y. (2011). Effect of heat-moisture treatment on the formation and physicochemical properties of resistant starch from mung bean (Phaseolus radiatus) starch. Food Hydrocolloids, 25, 1702e1709. Li, H. Y., Zhu, Y. Q., Jiao, A. Q., Zhao, J. W., Chen, X. M., Wei, B. X., et al. (2013). Impact
of a-amylase combined with hydrochloric acid hydrolysis on structure and digestion of waxy rice starch. International Journal of Biological Macromolecules, 55, 276e281. Lomova, M. V., Brichkina, A. I., Kiryukhin, M. V., Vasina, E. N., Pavlov, A. M., Gorin, D. A., et al. (2015). Multilayer capsules of bovine serum albumin and tannic acid for controlled release by enzymatic degradation. Applied Material Interfaces, 7, 11732e11740. Man, J. M., Yang, Y., Zhang, H. J., Zhang, C. Q., Wang, F. M., Gu, Y. P., et al. (2013a). Morphology and structural properties of high-amylose rice starch residues hydrolysed by amyloglucosidase. Food Chemistry, 138, 2089e2098. Man, J. M., Yang, Y., Zhang, C. Q., Zhang, F. M., Wang, Y. P., Gu, M. H., et al. (2013b). Morphology and structural characterization of high-amylose rice starch residues hydrolyzed by porcine pancreatic a-amylase. Food Hydrocolloids, 31, 195e203. rez, K. W., Díaz, P. O., & Simsek, S. (2011). Starch Maribel, O. M., Luis, A., Bello-Pe characteristics of bean (Phaseolus vulgaris L.) grown in different localities. Carbohydrate Polymers, 85, 54e64. Miao, M., Jiang, B., Zhang, T., Jin, Z. Y., & Mu, W. M. (2011). Impact of mild acid hydrolysis on structure and digestion properties of waxy maize starch. Food Chemistry, 126, 506e513. Miao, M., Li, R., Jiang, B., Cui, S. W., Zhang, T., & Jin, Z. (2014). Structure and physicochemical properties of octenyl succinic esters of sugary maize soluble starch and waxy maize starch. Food Chemistry, 151, 154e160. Mutungi, C., Rost, F., Onyango, C., Jaros, D., & Rohm, H. (2009). Crystallinity, thermal and morphological characteristics of resistant starch type III produced by hydrothermal treatment of debranched cassava starch. Starch/ €rke, 61, 634e645. Sta Ørskov, E. R., & McDonald, I. (1979). The estimation of protein degradability in the rumen from incubation measurement weighted according to rate of passage. The Journal of Agricultural Science, 92, 499e503. Ozturk, S., Koksel, H., & Perry, K. W. (2011). Production of resistant starch from acidmodified amylotype starches with enhanced functional properties. Journal of Food Engineering, 103, 156e164. O’Brien, S., & Wang, Y. J. (2008). Susceptibility of annealed starches to hydrolysis by amylase and glucoamylase. Carbohydrate Polymers, 72, 597e607. on, A. (2004). Origin of the Pohu, A., Putaux, J. L., Planchot, V., Colonna, P., & Bule limited a-amylolysis of debranched maltodextrins crystallized in the A form: a TEM study on model substrates. Biomacromolecules, 5, 119e125. Rizkalla, S. W., Bellisle, F., & Slama, G. (2002). Health benefits of low glycaemic indexfoods, such as pulses, in diabetic patients and healthy individuals. British Journal of Nutrition, 88, 25e62. Sajilata, M. G., Singhal, R. S., & Kulkarni, P. R. (2006). Resistant starch - a review. Comprehensive Reviews in Food Science and Technology, 5, 1e17. Shi, Y. C., Cui, X. M., Birkett, A. G., & Thatcher, M. (2006). Resistant starch prepared by isoamylase debranching of low amylose starch. US Patent Office, Pat., 7, 081e261. Shi, M. M., Chen, Y., Yu, S. J., & Gao, Q. Y. (2013). Preparation and properties of RS III from waxy maize starch with Pullulanase. Food Hydrocolloids, 33, 19e25. Shi, M. M., & Gao, Q. Y. (2011). Physicochemical properties, structure and in vitro digestion of resistant starch from waxy rice starch. Carbohydrate Polymers, 84(3), 1151e1157. Sun, Q. J., Gong, M., Li, Y., & Xiong, L. (2014b). Effect of retrogradation time on preparation and characterization of proso millet starch nanoparticles. Carbohydrate Polymers, 111, 133e138. Sun, Q. J., Li, G. H., Dai, L., Ji, N., & Xiong, L. (2014a). Green preparation and characterisation of waxy maize starch nanoparticles through enzymolysis and recrystallization. Food Chemitry, 162, 223e228. Valodkar, M., & Thakore, S. (2011). Organically modified nanosized starch derivatives as excellent reinforcing agents for bionanocomposites. Carbohydrate Polymers, 86, 1244e1251. Zeng, F., Chen, F. Q., Kong, F. S., Gao, Q. Y., Aadil, R. M., & Yu, S. J. (2015). Structure and digestibility of debranched and repeatedly crystallized waxy rice starch. Food Chemistry, 187, 348e353. Zhang, G., Ao, Z., & Hamaker, B. R. (2006). Slow digestion property of native cereal starches. Biomacromolecules, 7, 3252e3258. Zhang, H. X., & Jin, Z. Y. (2011). Preparation of resistant starch by hydrolysis of maize starch with pullulanase. Carbohydrate Polymers, 83, 865e867. Zhang, G. Y., Sofyan, M., & Hamaker, B. R. (2008). Slowly digestible state of starch: mechanism of slow digestion property of gelatinized maize starch. Journal of Agricultural and Food Chemistry, 56, 4695e4702. Zhao, X. H., & Lin, Y. (2009). The impact of coupled acid or pullulanase debranching on the formation of resistant starch from maize starch with autoclaving-cooling cycles. European Food Research and Technology, 230, 179e184. Zou, J. W., Zhang, F., Huang, J., Chang, P. R., Su, Z. M., & Yu, J. H. (2011). Effects of starch nanocrystals on structure and properties of waterborne polyurethanebased composites. Carbohydrate Polymers, 85, 824e831.