3.1 Structural comparison of native glucose polymers
The structural differences between three substrates were measured using SEC-MALLS-RI. As shown in Table 1, Mw of phytoglycogen, amylopectin and glycogen were 2.14×107, 3.74×107 and 0.53×107 g/mol, and Rz were 43.1, 128.2 and 29.4 nm, respectively. Both Mw and Rz were in the following order: amylopectin > phytoglycogen > glycogen. The Mw increased as the Rz increased for different sources of glucose polymer, indicating a positive relationship between Mw and Rz. Mw of phytoglycogen was lower than that of amylopectin, due to the absence of starch debranching enzyme in starch biosynthetic in cereal endosperm1. According to the previous literatures 1, 4, 7, amylopectin is composed of linear chains of α-1,4-D-glucose units connected through α-1,6 linkages (5–6%), with an average chain length of DP 13–24, whereas phytoglycogen is the amylopectin analogue in the endosperms from sugary-1 mutations of cereal grains with decreased activity of isoamylase-type starch debranching enzyme, forming linear chains with average chain length of DP 10–12 and 7–10% branch points. Also, the dispersed molecular density of phytoglycogen was approximately 10-fold than that of amylopectin 7, 23. Glycogen depicts the similar structural feature of phytoglycogen, however, there are two types of structural subunits known as α and β particles and larger clusters of α particles in a cauliflower-like structure is composed of small β particles 8. Moreover, the dispersity (D) was used as a measure of the breadth of the molar mass distribution and D were in the following order: amylopectin (2.25) > glycogen (1.47) > phytoglycogen (1.13), indicating that there was only monodisperse phytoglycogen arranged in chains of equal size. Similar observations for phytoglycogen from different sugary maize cultivars have been reported by Miao and coworkers 4.
Sample
|
Mn
(×107 g/mol)
|
Mw
(×107 g/mol)
|
Mz
(×107 g/mol)
|
D
|
Rz
(nm)
|
Table 1
Molar mass and radius of gyration of native phytoglycogen, amylopectin and glycogen.
Phytoglycogen
|
1.89 ± 0.05
|
2.14 ± 0.01
|
2.66 ± 0.07
|
1.13 ± 0.08
|
43.1 ± 0.7
|
Amylopectin
|
3.72 ± 0.10
|
8.37 ± 0.06
|
7.46 ± 0.04
|
2.25 ± 0.21
|
128.2 ± 2.3
|
Glycogen
|
0.36 ± 0.02
|
0.53 ± 0.05
|
0.75 ± 0.11
|
1.47 ± 0.12
|
29.4 ± 0.6
|
Mn, number-average molar mass; Mw, weight-average molar mass; Mz, z-average molar mass; D, dispersity, D was calculated as Mw/Mn; Rz, z-root mean square radius of gyration.
3.2 Acid hydrolysis
The hydrolysis profiles of acid-treated phytoglycogen, amylopectin and glycogen over 120 h are presented in Fig. 1. The time course of acid degradation of substrate took place in two stages. In the first stage (0–48 h), the degree of hydrolysis increased substantially up to higher than 70% due to rapidly degradation of glucose polymer, whereas the degree of hydrolysis of substrate incrementally increased to the limit values of approximately 100% at 120 h for the second stage. The similar trends were observed for three polymers, however, the degree of hydrolysis was in the following order under the same reaction conditions: amylopectin > phytoglycogen > glycogen, which may be related to the different fine structure of substrate. The differences in the unit chain composition, chain length, molar mass, particle size and molecular density of substrates might be attributed to the variation in acid hydrolysis profiles 14,17.
Combined chromatogram data from different substrate (Table 1), the degree of hydrolysis increased by the increased molecular size of glucose polymer. Sullivan and coworkers also reported that the larger particles in glycogen appeared to be degraded significantly more than the smaller one, suggesting that the α-1,4 glycosidic linkage resistance to acid hydrolysis compared to the protein-like links between particles 8. Moreover, the degradation rate of the terminal α-1,4 linkage was faster than the other α-1,4 linkage on the glucan chain, whereas α-1,4 linkage was hydrolyzed seven times as fast as the α-1,6 linkage at room temperature 14. On the basis of the above data, five types of acid-treated samples (phytoglycogen, amylopectin or glycogen subjected to acid hydrolysis for 5 min, 30 min, 2 h, 6 h, or 12 h, respectively) were selected to elucidate the kinetic and structure in subsequent experiments.
3.3 Molar mass and radius of gyration analysis
As shown in Fig. 2A, the chromatogram of phytoglycogen appeared as a single symmetrical narrow peak, indicating a homogenous polysaccharide as suggested by our previous study 4. Upon acid hydrolysis of phytoglycogen, the distribution gradually shifted to the smaller molar mass distribution region with significantly broadening. It was noteworthy that a shoulder appeared in the distribution curve in the early stage of acid hydrolysis (after approximately 30 min), due to the formation of small fractions. The bimodal distribution curve appeared after hydrolysis of 12 h, revealing the heterodisperse characteristic of the treated phytoglycogen. For the acid hydrolysis of amylopectin, in contrast, the distribution changed from bi-modal to mono-modal shape (Fig. 2B), which indicated that the larger fragments of amylopectin would be preferentially degraded to small ones. Li and Hu reported that the first-order kinetics models were applied to fit the evolution curve of starch chain-length and molecular size by acid hydrolysis treatment and the fast hydrolysis phase involved degradation of amylopectin long intra-cluster branches 16. This was related with the hydrolysis mechanisms: hydrogen ions prefers to hydrolyze the 2 or 3 glucose units away from the branching points of amylopectin long intra-cluster branches, and amylopectin short intra-cluster branches connecting the two neighboring double helices are less preferable for the acid hydrolysis. After acid hydrolysis, small peaks (DP < 12) and peak shoulders (DP > 12) were detected between the two main peaks of DP 13–15 and 25–27, which were suggested to arise from the branched amylopectin 18. Moreover, Powell et al. found that acid degradation occurred uniformly thought the particulates and larger molecules degraded more quickly 15. As shown in Fig. 2C for the acid degradation of glycogen, a broader eluting peak was observed after 5 min of acid hydrolysis, suggesting that glycogen was more heterogeneous and a fraction of larger particles was hydrolyzed into smaller particle during acid degradation, which was consistent with the previous observations 15.
Moreover, the variation of different average molar mass with acid treated time is shown in Fig. 3. A significant reduction in molar mass was observed during the course of acid degradation of Mn, Mw and Mz with the maximum peak shifting over time. The change of Mn was directly associated with the rate of reaction, however, there was a log relationship between molar mass and rate of reaction. Noticeably, Mn decreased more rapidly than Mw, whereas Mw decreased more rapidly than Mz in the early stages of hydrolysis. The greater degradation for amylopectin in the initial stage of acid hydrolysis was observed from the molar mass change of profile, compared with the phytoglycogen or glycogen as shown in Fig. 3. This might be related with the structural difference among the three substrates, resulting in varying small fragments formed during acid hydrolysis 21.
3.4 Kinetic modeling for depolymerization
To investigate the depolymerization kinetic of acid hydrolysis, the mathematical model was used to reflect the linkage cleavage of polymer by measuring the molar mass distribution with reaction time. As shown in Table 2, the molecular density (ρ) of phytoglycogen, amylopectin and glycogen were 266.42, 17.75 and 209.39 g/mol·nm3, respectively, which indicated that either phytoglycogen or glycogen had more tightly packed rigid structure with greater dispersed density, compared to the amylopectin with long chain length for the relatively flexible structure 4. According to Huang et al. 23, ρ of phytoglycogen was approximately 1000 g/mol·nm3, greater than that of amylopectin (40 g/mol·nm3), which were comparable with our data. After acid reaction, the value of ρ dropped substantially in the first 6 h, especially for phytoglycogen and glycogen. During the initial 5 min of acid treated glycogen, ρ was reduced to approach a half (from 209.39 to 107.41 g/mol·nm3), due to the formation of smaller particles (e.g. β particles or other degradation products) from glycogen degradation. This might be related with the chemical complexes exist between glycogen and binding protein under the “crowding-assembly” model 15. Prats et al. reported glycogen as a proteoglucan has three structural types that termed α-granule, β-granule and γ-particle 24. The α-granules are mainly found in liver and appear as aggregates of β-granules in a rosette-like pattern, while β-granules with molar mass of 106-107 Da have reported to exist in muscle with individual spherical structure containing several γ-particles. Also, the β particles have diameters varying between 10 nm and 50 nm, whereas the α particles have diameters as large as 300 nm, in a tetramer structure composed of small β particles 8. It was suggested that the binding protein in glycogen was hydrolyzed faster than the glycosidic bonds under the acidic condition. A similar phenomenon was confirmed in our study as depicted in Fig. 3 and Table 2.
Table 2 Determination of the fine structure and kinetics of degradation products from phytoglycogen, amylopectin and glycogen over time.
Time (min)
|
Phytoglycogen
|
Amylopectin
|
Glycogen
|
ρ
|
D
|
CDR
|
ρ
|
D
|
CDR
|
ρ
|
D
|
CDR
|
0
|
266.42
|
1.13
|
1.07
|
17.75
|
2.25
|
1.13
|
209.39
|
1.47
|
0.90
|
5
|
228.93
|
1.50
|
0.87
|
17.18
|
2.17
|
0.77
|
107.41
|
1.21
|
0.63
|
30
|
132.47
|
1.69
|
1.08
|
13.12
|
2.81
|
0.78
|
89.69
|
1.36
|
0.37
|
120
|
24.58
|
2.03
|
1.11
|
7.10
|
2.80
|
0.85
|
16.57
|
1.63
|
0.23
|
360
|
8.13
|
2.44
|
1.74
|
2.20
|
3.03
|
1.29
|
12.45
|
2.92
|
1.37
|
720
|
0.80
|
2.81
|
2.26
|
3.71
|
12.4
|
7.36
|
9.82
|
4.99
|
0.82
|
k (10-5 s-1)
|
3.45
|
6.13
|
0.96
|
ρ, molecular density; D, dispersity; CDR, combined dispersity ratio; k, rate constant.
ρ (g/mol·nm3)was calculated as Mw/Rz3.
D was calculated as Mw/Mn.
CDR was calculated as Mw2/MnMz.
D was defined as the broadness of the molar mass distribution and showed an increase with time, due to the formation of lower size fragments, as depicted in the Fig.2. Phytoglycogen had a high molar mass as well as broad molar mass distribution, and D increased rapidly from 1.13 to 2.81 during the course of acid reaction. The similar trends were observed for glycogen and amylopectin, due to diversified structural products after acid degradation process. Significantly, D of glycogen decreased from 1.47 to 1.21 during the 5 min of acid treatment, indicating the obtained monodisperse particle was associated with the smaller particles followed by decreasing the larger size particles. The combined dispersity ratio (CDR) was unity for a log-normal molar mass and the enrichment in the higher weight region led to lower value of CDR and vice versa 21. The original molar mass distribution was approximated by a lognormal curve with good accuracy (CDR at closer to 1). The CDR values for the degradation of the narrow phytoglycogen showed an increase with hydrolysis time.
Based on the simplified equation and experimental data, the value of α indicated the dependence of the reactivity of polymer on the molar mass and has been determined to be 1/2. A plot of 1000/Mn1/2 versus reaction time gave a straight line as shown in Fig. 4. The value of k for phytoglycogen, amylopectin and glycogen were calculated from the slopes to be 3.45×10− 5, 6.13×10− 5 and 0.96×10− 5 s− 1, respectively, which was consistent with the trends of hydrolysis profiles in Fig. 1. According to the previous studies 14, 16, the glucose polymer with a high molar mass was stable to acid degradation than smaller fragments, and linkages near the end of polymer chain had a higher reactivity than those at the center. Moreover, the branched structure played a key role on the reactivity of polymer: more branched implied more rigid macromolecules, which suggested the chains formed highly banched and packed dentritic structure for the higher integrity of phytoglycogen or glycogen, compared to amylopectin with relatively flexible structure and long internal chains 4, 23. Therefore, the above results might indicate that the reactivity of the linkages changes due to the composition of chain segment, since the mobility of chain was in the following order: amylopectin > phytoglycogen ≈ glycogen, and phytoglycogen or glycogen with relatively rigid structure was more resistant to hydrolysis than amylopectin. However, further studies of branch-chain lengths and glycosidic linkages of substrates as well as the acid concentration need to be conducted to address this issue, which certainly would help us to understand the fine structure in depth.
3.5 Particle size distribution analysis
The particle size distributions of acid-treated phytoglycogen, amylopectin and glycogen are shown in Fig. 5. The particle size distributions of native phytoglycogen, amylopectin and glycogen have been observed to exhibit the ranges of 25–122, 18–70 and 13–60 nm, respectively. The average particle size was in the following order: amylopectin > phytoglycogen > glycogen. After acid hydrolysis, the distribution gradually shifted to the smaller particle size distribution. For instance, the particle size distributions were 5–18, 2–10, 3–15 nm for acid treated phytoglycogen, amylopectin and glycogen with 720 min, respectively, which was comparable with the obtained molar mass distributions as shown in Fig. 2. More specifically, there was a little narrower peak observed for the acid-treated glycogen with 5 min reaction, compared to the native glycogen, which was in accord with the results of D in Table 2. This was suggested that formation of smaller particles (mainly β particles) in the early stage of acid degradation due to binding protein hydrolysis in glycogen.
3.6 1H-NMR analysis
To further characterize the structural properties of acid-treated phytoglycogen, amylopectin and glycogen, their concentrations of α-1,4 and α-1,6 bonds were analyzed using 1H NMR spectroscopy. This approach can be used to determine the degree of branching of glucan dendrimer polymer, including phytoglycogen, glycogen and amylopectin. As shown in Table 3, the percentage of α-1,6 linkages of native phytoglycogen, amylopectin and glycogen were 7.6%, 5.2% and 6.5%, respectively, which indicated that the following order of phytoglycogen > glycogen > amylopectin. Similar observations for glucan dendrimer polymers have been reported by BeMiller and Whistler 1. According to the previous studies 4, 25, phytoglycogen has been proposed to bear a resemblance to the glycogen isolated from animal sources. Phytoglycogen is made up of α-1,4-linked glucose units, forming linear chains with 7–10% branch points introduced by α-1,6 linkages that are relatively evenly distributed throughout the particles, and is primarily composed of internal short B-chains attached by external short B- and/or A-chains, with no long B chains 7. Also, Yun and Matheson reported that glycogen has A:B chain ratio (where A chains contain only α-1,4 linkages and B chains also contain α-1,6 linkages) of < 1 and amylopectin ratios of > 1, which led to average frequencies of substitution of B chains over the whole molecule of < 2 for the glycogens and > 2 for the amylopectins 26. After acid hydrolysis, the 1H NMR spectra showed an decrease of α-1,6 linkages in the acid treated samples, which was related with the acid hydrolysis of glucosidic linkages in glucan chain. Especially, greater degradation was observed for phytoglycogen from 7.6–2.3% during acid hydrolysis course of 720 min, which might be due to larger particle size of phytoglycogen as shown in Fig. 5. The branched structure of glucan polymer played a key role on the acid degradation: more size implies larger surface, resulting in rapid hydrolysis of glucan chain with more time, comparable with the obtained molecular density as shown in Table 2. Moreover, there was no change for percentage of α-1,6 linkages in glycogen in the first 5 min, which might be related with the special aggregation structure of glycogen containing degradable protein as described by Tan and coworkers 27. The above results suggest that mild acid acted on glucan chains and produced sample with shorter branch chains and less α-1,6 linkages.
Table 3 Concentrations of α-1,6 linkages of acid-treated phytoglycogen, amylopectin and glycogen.
Time
(min)
|
Percentage of α-1,6 linkages (%)
|
Phytoglycogen
|
Amylopectin
|
Glycogen
|
0
|
7.6±0.3
|
5.2±0.5
|
6.5±0.5
|
5
|
7.3±0.5
|
4.8±0.1
|
6.5±1.0
|
120
|
5.8±0.2
|
3.7±0.2
|
5.9±0.2
|
720
|
2.3±0.6
|
1.9±0.3
|
2.8±0.2
|
3.7 In-vitro digestion
Table 4 shows the enzymatic digestibility of acid-treated phytoglycogen, amylopectin and glycogen when subjected to the Englyst assay. According to the rate and extent of digestibility, the glucose polymer is divided into rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch (RS), which are the three consecutive nutritional fractions divided by reaction time and related to the physiological effect of processed food after consumption 22. The results showed that RDS, SDS and RS were 74.7%, 12.2% and 13.1%, 72.4%, 10.7% and 16.8%, 69.9%, 12.9% and 17.2% for phytoglycogen, amylopectin and glycogen, respectively, consistent with previous studies 4,7. It has been reported that the enzymatic digestibility of glucan is influenced by source, particle size, ratio of α-1,4 to α-1,6 bonds, molar mass and branch chain length 28, 29. This suggests that the total value for RDS and SDS of amylopectin was lower than that of phytoglycogen or glycogen, which was due to its lower α-1,6 bonds and longer branch chain length as well as larger particle size. Pazur and Ando observed that hydrolysis of α-1,6 bond of glucose polymer was the rate-limiting step for the amylolytic degradation 30, which revealed that the enzymatic hydrolysis rate of α-1,6 bond was approximately 30 folds lower than that of α-1,4 linkage. Also, there was a parabolic relationship between amylopectin molecular structure and SDS, which meant amylopectin with a higher amount of either short chains (DP < 13) or long chains (DP ≥ 13) had a higher content of SDS 31,32. As illustrated in Table 4, the percentage of RDS was significantly increased in all of acid-treated samples, accompanying with decreasing the content of SDS. After acid hydrolysis, RS were composed of slightly difference, except for amylopectin with 720 min treatment, which might be attributed to the difference of branching patterns. Thus, the fine structure of glucose polymer substrate influencing the in vitro glucose release was a crucial factor for enzymatic digestibility, and the treated sample with more branch points, shorter chains and smaller size had a higher SDS and RS content. According to the recent review of Miao and Hamaker28, the major intrinsic factors affecting starch digestibility include the botanical source, granule size, crystal architecture, ratio of amylose to amylopectin, amylopectin fine structure, and surface and interior characteristics of the starch granule. The smaller size of glucan samples have higher amylolysis rates than the larger counterparts, which is related with the increased surface-to-volume ratio of smaller particle for enzyme binding to the substrate, leading to a proportional increase in digestibility for acid-treated amylopectin. Also, there was a higher proportion of α-1,6 linkages in starch molecules, which are less efficiently degraded by amylase, resulting in low digestion properties. For the acid-treated glycogen, RS content increased by the increased hydrolysis time, which might be related with more α-1,6 linkages and larger particle size as well as some small product molecules with amylase inhibitory activity. Further studies need to be conducted to address this issue, which certainly would help us to understand the more resistant fractions of acid-treated glycogen in depth.
Table 4 In vitro digestion of acid-treated phytoglycogen, amylopectin and glycogen.
Time
(min)
|
Phytoglycogen
|
Amylopectin
|
Glycogen
|
RDS(%)
|
SDS(%)
|
RS(%)
|
RDS(%)
|
SDS(%)
|
RS(%)
|
RDS(%)
|
SDS(%)
|
RS(%)
|
0
|
74.7±1.3
|
12.2±0.5
|
13.1±1.0
|
72.4±0.8
|
10.7±1.1
|
16.8±0.2
|
69.9±0.5
|
17.9±1.5
|
12.2±1.2
|
5
|
74.8±0.8
|
12.0±1.2
|
13.2±0.9
|
76.7±1.5
|
10.3±0.2
|
13.0±1.4
|
70.7±1.1
|
10.7±0.9
|
18.6±1.0
|
120
|
79.0±0.5
|
8.3±0.6
|
12.7±1.2
|
80.1±1.2
|
7.7±0.8
|
12.2±0.7
|
73.8±1.0
|
6.9±0.6
|
19.3±1.5
|
720
|
84.1±1.1
|
4.0±0.6
|
12.0±0.3
|
89.0±1.4
|
4.0±0.7
|
7.1±0.4
|
75.4±1.6
|
4.8±0.4
|
19.8±0.7
|
RDS, rapidly digestible starch; SDS, slowly digestible starch; RS, resistant starch.
3.8 Structural model of acid degradation.
The structural change of phytoglycogen, amylopectin and glycogen subjected to acid degradation was deduced and the possible model is present in Fig. 6. According to the previous studies 4, 9, 12, both phytoglycogen and amylopectin are the highly branched α-D-glucan naturally occurring in plants. The starch biosynthesis reactions are coordinately catalyzed by the combined action of starch synthase, branching enzyme and debranching enzyme to form amylose and amylopectin in the amyloplasts 1. In the debranching enzyme-deficient mutant of sugary-1 endosperm, phytoglycogen is formed as an immature precursor of amylopectin with more highly branched structure. A mathematical model has been developed for describing the Whelan structure of phytoglycogen 33. The molecule has a spherical shape that is organized into concentric tiers. Every B chain is in the inner tier and every A-chain is in the outer tier. Each A- or B-chain has 12–14 glucose residues and there are a maximum of 12 tiers in the molecule, which means there is a maximum of about 53,000 glucose residues and 2,100 non-reducing ends. Within the tiered structure, the actual size is limited by the space available at the periphery for further branching. The differences in molecular architecture within the phytoglycogen led to a change in density change from the internal (950 g/mol·nm3) to external (1,600 g/mol·nm3) regions, different from the density of homogeneous amylopectin with approximately 60 g/mol·nm3 7, 23. Also, the external region of the phytoglycogen was reported to be about 3.3 nm thick. Moreover, glycogen is the intracellular major glucose reserves in the animal tissues, and has been identified to bear a resemblance to phytoglycogen from plant origin 8. In the Krisman model, there is a protein core surrounded by several tiers of branch points, with crowding in the outer layers 34. Glycogen comprises complex α particles made up of smaller β particles, ranging from 20 to 300 nm in diameter. The two mechanisms in glycogen synthesis for stopping chain growth were proposed: branching enzyme dominated chain growth in the innermost region of complex branched polymers with low molecular density, while steric hindrance (crowding) dominated chain stoppage in the high-density outer region 35. Also, the protein glycogenin was confirmed as binding agent joining β particles together into large α particles 27. Overall, phytoglycogen or amylopectin appeared to be held together only by glycosidic linkages, whereas α-particles in an animal-based glycogen were held together by a combination of covalent and non-covalent links involving a protein.
In combination with the results of fine structure and particle size distribution (Table 3 and Fig. 5), amylopectin with a higher particle size and lower density was hydrolyzed faster than phytoglycogen or glycogen counterparts, suggesting a less branch and relatively flexible structure tends to give higher rate of hydrolysis degradation. In the initial stage of acid hydrolysis (approximately 0–30 min), the protein in glycogen α particles was more acid-labile than the glycosidic linkage under mild conditions, resulting in the smaller β particles, which were consistent with of our observations of molar mass and particle size distributions. In the second stage, the degradation of glycogen fitted the uniform degradation model, depicting the non-preferential linkage cleavage as observed for the similar course of acid-treated phytoglycogen. A combination of structural data and synthesis mechanism showed that acid hydrolysis process fitted the successively depolymerization of Krisman or “crowding-assembly” and “crowding/budding” models of glycogen. For phytoglycogen or amylopectin, the acid degradation fitted the depolymerization of Whelan or “crowding/budding” model, different from structural changes of glycogen. Moreover, the particle size distribution of phytoglycogen decreased uniformly with hydrolysis time as shown in Fig. 5.