3.2. Rheological properties
The dynamic rheological properties storage modulus (
G′) and loss modulus (
G′′) are presented as a function of frequency in
Figure 2 and
Table 1, there were significant differences among the five mashed potato samples (with and without protein addition) with respect to frequency dependence during rheological measurements. In each case, the modulus versus frequency curves for the different samples exhibited no cross-over within the range of frequency accessed (
Figure 2),
G′,
G′′ and complex (
G*) increased while complex viscosity (
η*) and dynamic viscosity (
ή) decreased with increasing frequency. All the values of rheological parameters
G′,
G′′,
G*, loss tangent (tan δ),
η* and
ή increased with the addition of both PPI and SPI in the Yangyu jiaotuan, and the values increased with the increasing of levels of addition except the loss tangent.
The G′ of control on the zero-day was about 12,000 Pa, remained the same on the first day, then increased to about 17,000 Pa on the third day, and increased to about 22,000 Pa on the 5th day, which indicates the Yangyu jiaotuan became hardened due to starch retrogradation during the storage. The values of Yangyu jiaotuan made with 5% plant protein (both PPI and SPI) are close to the control at zero-day, and during the storage of 1 day, 3 days, and 5 days, it has very similar characteristics to the control. At the end of even the 5th day, the Yangyu jiaotuan made with 5% SPI addition had the lowest viscosity values of among all, it was even lower than the control which indicates the palatability wise, the characteristics wise. The values of rheological parameters increased significantly when 5%PPI, 10%PPI, or 10% SPI was added, Yangyu jiaotuan made with 10% PPI gave the highest viscosity (all rheological parameters) in combination with potato, it was very different from the control, the formulation has very strong gel already.
Rheological techniques can also be used to monitor the development of viscoelastic properties of starch during retrogradation [
18]. In this study, during the refrigerated storage at 4
oC, the values of
G′,
G*, and
η* increased significantly with the prolonging of storage time from 0 day to 5 days, while the values of
G′′, and
ή did not change significantly. The values of tan δ decreased, exhibited behavior contrary to
G′,
G′′,
G*,
η*, and
ή, indicating the formation of stronger network structures in retrograded pastes.
Table 2 illustrates the extent of change in
G′,
G′′, and
η* of percentage. For the control sample, the value of
G′ after 5 days has increased 77.27%, while it was lower for the sample containing 5% PPI, it was 45% increase. The extent of change in
G′′ and
ή in terms of percentage show absolutely the same pattern as the
G′ values, this means that protein has helped the sample to prevent the retrogradation because it didn't harden. Usually, consumers like to eat the soft, so we can keep Yangyu jiaotuan soft during the storage period by adding plant proteins. Moreover, the value of
G′ after five days has increased 29.46% and 53.35% for the Yangyu jiaotuan containing 10% PPI and 10% SPI, which indicate that with a high extra protein addition, PPI is more effective in preventing the starch retrogradation and ultimately obtaining more smooth, consistency or a soft consistency over the storage period.
Although, the rheological parameters have already giving a lot of information, a lot about different proteins, and how they are affecting starch retrogradation. But to find out the mechanism by which these proteins are affecting, because that'll make it more interesting, rather than leaving it on a superficial, and how we can make use of that. Whether there is any molecule level interaction among them in terms of the bonding, or they're just helping to absorb the water which comes out of starch due to syneresis. What is the mechanism? It worth further study.
The Pearson correlation coefficients for the relationships between textural and rheological properties are presented in
Table 3. Hardness, Gumminess, Chewiness and Resilience showed a positive correlation with
G′ (
r = 0.658, 0.680, 0.659, 0.692, respectively), whereas rheological properties such as G0, G00 and peak viscosity showed a positive correlations (r = 0.690, 0.847, 0.736, respectively).
The correlation between these texture parameters and G′′ and G* shows the same pattern, because gumminess, chewiness, and resilience showed a strong positive correlation with hardness, while G′′, G*, η*, and ή also has a strong positive correlation with G′.
3.3. Starch hydrolysis (%) and estimated glycaemic index
The starch hydrolysis (%) of Yangyu jiaotuan during in vitro oral-gastro-small intestinal digestion is shown in
Figure 3. During the simulated oral digestion phase (O and G0), α-Amylase is generally well integrated within the food bolus and continues to release some glucose, starch hydrolysis (%) observed during the oral digestion process ranged from 2.27% to 14.60%. During the simulated gastric digestion phase (G0 and G30), the starch hydrolysis (%) of all samples were barely changed indicating the activity of α-Amylase is very low due to the extremely low pH of 2 (
Figure 3). During the simulated small intestinal digestion, the starch hydrolysis (%) of all samples increased rapidly, especially at the initial phase. From
Figure 3, it was obvious that the starch hydrolysis (%) of all samples decreased with the extension of refrigerated storage time from 0 to the 5
th day, the gradually decreasing starch hydrolysis (%) of the 0-day (76.34%), 1-day (72.17%), 3-day (65.09%) and 5-day (57.19%) of the plain Yangyu jiaotuan (control) at the I15 point indicated that refrigerated storage resulted in the starch retrogradation process and subsequent the rate of enzymatic digestion, which is consistent with our previous study [
14].
Equilibrium starch hydrolysis percentage (C
∞), areas under hydrolysis curves (AUC), hydrolysis index (HI), and estimated glycaemic index (
eGI) were estimated by fitting a first-order equation model according to the reported method [
16]. The HI of fresh prepared plain Yangyu jiaotuan was 129.84 while the white read was used as a reference. The HI and the
eGI of all the samples decreased with increasing refrigerator storage time indicating storage affected the rate of enzymatic digestion (
Table 4). The experimental C
∞ of 5% PPI and 10% PPI samples after 5-days refrigerated storage were significantly lower than the values of other samples, while the differences of values of other samples are not significant. For the same refrigerated storage time of 0, 1, 3, and 5 days, the higher the amount of protein added, the lower the HI and
eGI values. The 10% PPI after 5-days of refrigerator storage had the lowest HI (41.76) and
eGI (62.63) values, which is significantly lower than the other values of the control and 5% PPI. Factors that influence the kinetics of starch digestion are the nature of starch, physical form, protein and lipids interactions, presence of antinutrients enzyme inhibitors, and food processing [
19].
Figure 3 and
Table 4 clearly show that the newly developed product with extra plant protein has lower starch hydrolysis or estimated glycaemic index than the control sample implying the gel network made with globular protein granules could act as a physical barrier impeding amylase accessibility to the encapsulated starch, in a similar manner to that seen with cell walls. The protein gel network appeared to hinder the free access of amylolytic enzymes to the encapsulated starch, therefore providing a slow and extended-release of glucose during
in vitro digestion. This means the high protein Yangyu jiaotuan are disassembled and release nutrients in the digestive tract at a slower rate with greater satiety as opposed to plain mashed potato.
The ultimate vision of this study is not to make a replica of natural foods, but rather to be inspired by structure-functionality linkages encoded in them, to enable the design of nature-like food systems with similar or enhanced functionality. We develop a category of BPF product of high protein Yangyu jiaotuan that can be tailored to allow their uses in specific health-promoting applications to help prevent or combat chronic diseases include lowered caloric content and prolonged satiety for obesity management, slow and sustained release of glucose in the small intestine and delivery of resistant starch to the large intestine for decreased risks of diabetes and improved colonic health.
3.4. Microstructural
Microphotographs of Yangyu jiaotuan are shown in
Figure 4. As shown by the microphotographs of a light microscope (
Figure 4A, B, and C), large size particulars in blue and small size particulars in golden can be distinguished, they refer to starch (stained by 1% iodine indicator) and globular protein, respectively. A fairly amount of golden granules can be found in the Yangyu jiaotuan made with 5% PPI (
Figure 4B) and 5% SPI (
Figure 4C), the morphologies characteristics such as color and shape were very similar, but the particle size of SPI was relatively higher than that of PPI.
After the addition of extra plant protein, two different types of gel networks were formed: fine-stranded and coarse networks [
12]. The absence of adhesion between starch matrix should be disregarded as they are not features of the different samples but a problem of sample preparation. A large amount of liquid was introduced during the staining of the light microscope. A strong gel network of the starch matrix was formed in Yangyu jiaotuan, which can be confirmed by the results of the CLSM images (
Figure 4G, H, and I). The opaque coarse gels network defined as the white aggregate was made with starch matrix, Yangyu jiaotuan with and without extra plant protein addition consist mainly of a continuous phase (amylose/amylopectin matrix) due to the disruption and complete solubilization of the potato starch granules by heating.
Another opaque fine-stranded gel network was particulate gel made with globular protein granules [
20]. In both PPI (
Figure 4C) and SPI (
Figure 4D) Yangyu jiaotuan, a protein network structure can be seen composed of protein aggregate clumps and small protein clusters, which are distinguishable from the starch matrix. Tseng et al. also observed that SPI gels exhibited a particulate porous network structure [
21], Alvarez et al. reported two different types of gel network in mashed potatoes enriched with SPI [
12].
To achieve a better understanding of the starch matrix of Yangyu jiaotuan, fresh prepared Yangyu jiaotuan was washed with plenty of water and followed by freeze-drying, the microstructure of the systems was studied by SEM (
Figure 4D, E, and F). Individual cells were successfully isolated and captured, the size of freeze-dried potato cells ranged between 100 to 200 microns, which is much smaller than the potato parenchyma cells ranged between 100 to 1000 microns [
11]. The starch matrix is tightly packed within the cell wall polymer network, and the folds on the surface of microspheres can explain the dehydrating effect of freeze-drying on the shrinking cell volume. Micrographs also revealed the presence of cell wall cementing materials as well as cell fr aments are embedded in the continuous solubilized starchy matrix (
Figure 4D and F), which was consistent with the previous report [
12]. The native starch granules absorbed abundant cell fluid and gelatinized during hydrothermal cooking, followed by the separation of intact cells (instead of rupturing) during Yangyu jiaotuan processing due to the drastic mechanical shearing. Once there has been a substantial level of cell separation, it becomes rather difficult to fracture individual cells by any mechanical means [
22]. Berg, Singh, Hardacre et al. and Tydeman et al. also reported that hydrothermal processing of plant foods (e.g. legumes and carrots) and subsequent mechanical shear applied within the resulting puree induce separation of intact cells without breaking them open [
23,
24].
During the simulated oral digestion and simulated gastric-small intestinal digestion, microstructural changes of mashed potato, mixed with pea protein were also revealed by SEM (
Figure 5). After 2 minutes of oral digestion (
Figure 5A), the morphology of the starchy matrix did not change compared with that of freshly prepared Yangyu jiaotuan (
Figure 4D and E). Similarly, there was no obvious change after 30 minutes of simulated stomach digestion (
Figure 5B), this observation was attributed to the absence of amylases in the gastric juice with the minimal hydrolysis observed being attributed to acid pH [
25]. During the initial phase of simulating small intestine digestion, the starchy matrix shrank to become smaller and the cell wall polymer network increased (
Figure 5C). After 10 minutes of simulating small intestine digestion, a large number of cell wall polymer network fragments were reminded (
Figure 5D). As the digestion process progresses, the starch matrixes are nearly invisible, leaving only the cell wall polymer network fragments and the pea protein particles (
Figure 5E),
in vitro starch digestion assays combined with SEM techniques demonstrated that gelatinized starch, which is tightly packed within biopolymer matrices in cooked starch matrixes, was enzyme-digested in a layer-by-layer fashion (
Figure 5C2), this means that digestion progressed from the outer towards the center of the starch-entrapped microspheres [
1].
Figure 5F is a plain mashed potato without additional plant protein, no starch matrix could be found after 120 minutes of simulated small intestine digestion, and all that remained were cell wall polymer networks that could not be digested by amylase.
3.5. Particle size distribution and thermal properties of Yangyu jiaotuan digesta
The particle size distribution for Yangyu jiaotuan digesta with 5% PPI after 3-days of refrigerated storage are shown in
Figure 6. The bimodal peaks of the particle size distribution curves show that the newly developed Yangyu jiaotuan contains two different sizes of particles, the smaller particles corresponded to PPI (~30 μm), which is consistent with the CLSM and SEM results (
Figure 4I and
Figure 5E2). Our previous study revealed that plain cooked potato tuber after simulated digestion also contains some small particles sized between 15.2-35.1 μm [
14], which indicated that the small particles in the Yangyu jiaotuan also contain some starch granules [
26], which can be confirmed by the volume of large particles represented in Yangyu jiaotuan (~10%) was much greater than that of 0.5–1.5% represented in plain cooked potato. The larger particles in Yangyu jiaotuan corresponded to the fragments of a gel network of the starch matrix [
26], which is consistent with the SEM results (
Figure 5). The results also show that the integrated volume of the small particle sizes increased and the volume of the large particle sizes decreased as the digestion time extended, indicating that the fragments of starch matrix became smaller and smaller during the prolonged digestion.
The results about differential scanning calorimetry (DSC) thermogram of Yangyu jiaotuan digesta samples during the simulated oral-gastric-small intestinal digestion were showed in
Figure 7. The peak temperature (
Tp) of O2 and G30 were found to be 75.47 and 75.75
OC respectively, the endothermic peaks reduced in samples as the simulated digestion time extended, the samples of I10, I10, and I120 did not show endothermic peaks, indicating that the crystalline region was gradually destroyed [
27]. Besides, starch is quickly digested in the simulated small intestine, in particular the I120 sample, there is no starch matrix residue. These results were inconsistent with those of starch hydrolysis (
Figure 3) and SEM (
Figure 5).