2.1. Proanthocyanidins preparation
The catechin standard curve was taken as abscissa by the concentration of catechin standard solution, and the absorbance was taken as ordinate. The Fig.1 show the results for the regression equation : Y=0.001191X + 0.00123, R2=0.99988. The final extraction rate was 5.29 % and the relative deviation was 0.638 %.
The red curve was the HPLC-FLD chromatogram containing Catechin/epicatechin, Procyanidin dimer and Procyanidin tetramer, black curve was the HPLC-FLD chromatogram of redissolved proanthocyanidins after lyophilisation in Fig.2. From chromatograms and references, the main components of proanthocyanidins extracted by this method were Catechin/epicatechin, Procyanidin dimer and Procyanidin tetramer, and their ratio was 5:31:4.
2.2. Protein purity and particle size
The Biuret Method was used to detect the current purified protein, and the measured purity result was 90.08±2.27%, which was greater than or equal to the purity of some proteins studied6, 7, so the experiment was feasible in terms of protein purity. In this experiment, the freeze-dried protein was manually ground. The reason was to prevent mechanical grinding from transforming, degrading, or even destroying the internal groups and disulfide bonds between the proteins, which made the gel structure unstable8, which could affect the experimental results. In this experiment, mortar was used for grinding, and the protein particles after grinding were tested by DLS. DLS results show that the distribution of MP was relatively wide (2296-3592 nm) and the average particle size was larger (3007±18 nm), reflecting the uneven dispersion of MPs (Fig. 3).
2.3. Texture characteristics
Texture was closely related to sensory characteristics, physicochemical properties, and processing characteristics of gelatinous products. Hardness, chewiness and springiness were the important indicators for texture characteristics, because they directly affect the quality of gelatinous products.
In the Fig.4, category 0 was the blank contrast, and category 1-4 was 0.05, 0.10, 0.15, 0.20 g/kg NaNO2, abbreviated as C1, C2, C3, C4, respectively. Category 5-9 was 0.05, 0.10, 0.15, 0.20, 0.25 g/kg PFGS, referred to as C5, C6, C7, C8, C9, respectively. a was the data map of springiness, b was the data map of chewiness, c was the data map of hardness
It could be seen from Fig.4-a that springiness of the gel increased with the increase of NaNO2 and PFGS addition, but the springiness of the 0.20 g / kg proanthocyanidins group (PG) was only 8.3587 % ± 0.657 % higher than that of the NaNO2 group. In addition, Fig.18 showed that through the sensory evaluation of 300 random people, 96.832 % believed that elasticity did not change. It could be seen from the Fig.4-a that after 20 days of storage at 4℃, the springiness decay rate of the original anthocyanin group was lower than that of the NaNO2 group, and the decay rate was 13.574 % lower than that of the NaNO2 group. It was believed that the decrease of springiness was related to the loss of free water and the destruction of disulfide bonds in the gel during 20 days. It could be seen from Fig.8 that the volume measured by SPRS also showed that the gel had a certain degree of shrinkage and collapse. However, from the data, the gel collapse rate of the PG was slower than that of the blank contrast group. This may be due to the polymerization of C-ring in PFGS with C-C and C-N bonds in gel particles during the heating process of gel formation, which strengthens the interaction between bonds in gel.
The chewing force data in Fig.4-b showed that the chewing force of the gel in the NaNO2 group increased slowly with the increase of NaNO2 content. The masticatory force of the PG at 0.05 g / kg was lower than that of the blank contrast gel. Considering that the adhesiveness and cohesion of the gel formed were insufficient when the concentration of PFGS was too low, and the influence on the disulfide bond and the amide bond was unstable, which led to the lower masticatory force at 0.05 g / kg. However, with the increase of the amount of the original pigment, it could be seen that the chewing force had a very rapid growth trend. At the same time, the respondents also obviously feel that the gel chewing force of the original pigment group at 0.20 g / kg had been greatly strengthened, and the touch was more like rubber.
With the passage of time, the decline rate of chewing force in the NaNO2 group was roughly the same and the decline was slow. The chewing force in the PG at all concentrations decreased rapidly with time. It could be seen that PFGS could play an antioxidant and stabilizing role in gel formation, but PFGS were easy to polymerize and had too many oligomeric forms, resulting in the molecular groups of PFGS and gel formation were very different. As time goes by, different molecular groups turn into new molecular forms due to oxidation, resulting in a softening of the gel layer outside the gel. When the gel of the 20-day PG was sliced, it was found that the gel surface was soft and crisp, but the central part still showed gel properties. This was consistent with the observation trend of Raman microscope. It could be seen from Fig.4-c that with the increase of the concentration of the original pigment group and the NaNO2 group, the hardness of the formed gels increased, and with the passage of time, the hardness of the gels still increased, which was directly related to the water loss of the gels. The free water content of the two groups decreased significouldtly on 20 days, and the gels showed hard and brittle properties.
In summary, it could be seen that when the original pigment was 0.25 g / kg, the gel did not form bulky elastic substances, but formed flocculent condensation substances. The substances in this state did not had gel properties, and the detection also showed data different from the gel, which was corresponding to the following infrared characterization. When the original pigment was 0.25 g / kg, the data of formed substances were disordered. At the same time, it was found that the parallelism of PG was more unstable than that of NaNO2 group in the process of texture detection. It was analyzed that during the heating process of gel formation, different proanthocyanidins oligomers or polymers were formed, and different polymers had different effects on S-S, which made the experimental stability of PG lower than that of NaNO2 group. This could be seen from the experimental error.
2.4. FTIR analysis
FTIR was applied to further investigate the intermolecular interactions in Protein- Proanthocyanidins matrixes. As shown in Fig.5 the broad band centered at 3000-3600 cm−1 was assigned to O-H and N-H groups, implying the potential for hydrogen-bonding interactions。This means that with the addition of PFGS, O-H and N-H groups in the gel force increased,
Compared to blank contrast, the stretching vibrations of O-H and N-H groups appeared at 3000-3500 cm−1, revealed enhanced intensity profiles for 0.15% and 0.20% PFGS, which suggesting that the intensities of inter- and intra-molecular hydrogen bonds were enhanced. This was a favorable change in affecting affinities between proteins. The strong hydrogen bond potentials in the MPs surface contributed to the stability and order between proteins, and accelerated the process of rearrangement and aggregation. The change was then noticeably reflected in 0.20% PFGS which had a minimal particle size because of exposure of internal functional groups, or selfregulation of secondary structures within ground MPs due to that polymerization of PFGS during the formation of gel.
LF-NMR had been widely used to evaluate the distribution and mobility of different fractions of water molecules in a gel system. According to Fig.6-7, the T curves were well described by three separate peaks centered at approximately 10-70 ms, 10-500 ms and 500-3000 ms, revealing bound water, immobilized water, and free water, respectively. Among them, immobilized water, which reflected the trapped water molecules within the three-dimensional network, dominated the gel systems.
When H atom was on different molecules or in samples under different physical states, its relaxation time will be different. It could be seen from the Fig.6 that the relaxation time of three kinds of water in the gel added with PFGS at the 0th day of gel formation was significouldtly delayed. The addition of PFGS enhanced the stability of the gel. The delay in relaxation time proved that PFGS played a role in strengthening the water molecules of the gel. The reason may be that some gel molecules formed a spatial structure with more bound water with PFGS during polymerization, or that the reaction of PFGS with a large number of hydroxyl groups in the formation stage of the gel made H atoms more exposed.
Fig.7 and Table 1-5 showed the detection diagrams of five samples over time. It could be seen that only the relaxation time of T1 and T2 was advanced again in the fifth day and later detection. The reason may be that the hydroxyl of PFGS and gel molecules formed a more stable bound water under the action of air oxidation. In other words, PFGS induced the conversion of free water to immobilized water. Therefore, water mobility in the PG was somehow restricted. This phenomenon may be attributed to the well-aggrega.
It could be seen from the Fig.8 that on day 0, although the parameter was set as 1cm × 1cm × 1cm, the volume measured by SPRS decreases. Therefore, it was believed that the gel formed after the addition of PFGS had a closer spatial structure, and the gel density was also larger. Water molecules were not easy to volatilize, and the water retention was stronger. Before 10 days, the gel volume was positively correlated with the amount of PFGS added. After 10 days, the gel volume decreased rapidly with the addition of 0.15 and 0.20 g / kg PFGS, but still higher than the blank contrast. Therefore, it could be inferred that at the concentration of 0-0.2 g / kg PFGS, the water retention of low-concentration gels was more stable over time, and high-concentration PFGS had better water retention in the short term, but the water retention decreased in the long term.
2.6. Raman spectroscopy
Recently many studies had used Raman spectroscopy to reflect information about the secondary and tertiary structure of protein gel. The gel properties were related to changes of the secondary structure9. Generally, the Raman spectral bands of amide I (1600-1700 cm-1) and amide III (1200-1300 cm-1) were used for measuring the secondary structure10, 11. The MPs gel of high percent of α-helix, β-sheet, random coil and β-turn could be severally appointed at 1650-1658 cm-1, 1665-1680 cm-1, 1660-1665 cm-1 and 1680-1690 cm-1 12, 13.
Fig.9 and Fig.10 showed Raman spectra of gels treated with different concentrations PFGS. The bands of amide I centered at 1652 cm-1 and the bands of amide Ⅱ centered at 1234 cm-1(Fig.11 and Fig.12) were fitted through Gaussian curve and Fourier self-deconvolution (FSD) to analyze the structure of the protein. The band of 1652, 1663, 1674, and 1683 cm-1 respectively indicated α-helix, random coil, β-sheet and β-turn14. 1235 ± 10 cm-1 wavelength in the amide III region showed β-folding. The wavelength of 920-1180 cm-1(Fig.13) was N-Cα-C skeleton stretching vibration, which was classified as amide mode by some authors. Wavelength 1400-1500 cm-1 was the CαH2 bending mode of glycine or serine and wavelength 1509-1592 cm-1(Fig.14) was the amide II region
On the 0th day of gel formation, the addition of PFGS was positively correlated with the strength range of N-Cα-C skeleton stretching vibration, which was most likely due to the large amount of C=O, hydroxyl or benzene ring in Proanthocyanidins that enhanced the Cα-C strength of certain amino acids in the gel. However, with the passage of time, the strengthening effect of PFGS gradually weakened, and the higher the concentration was, the stronger the strengthening effect at the beginning was, and the faster the strengthening effect decreased with time.
It could be seen from the figure that β-sheet of amide 1 band 1673.892 nm-1(Fig.15) and amide II band 1234.649 nm-1 formed gel on the 0th day, β-sheet increased with the increase of concentration. This was probably because the polymerization of PFGS in the formation process takes away the charge of some amino acids in the gel. With the decrease of the same charge and the increase of the different charge, the β-sheet formed between the peptide chains becomes stronger and more stable. But with the passage of time, especially in 5-10 days, β-sheet had a rapid downward trend, after 10 days continue to decline slowly, may be the gel hydrogen bond or S-S was oxidized broken, resulting in gel instability.
As could be seen from the Fig.11, α-helix had little effect after adding PFGS The reduction of the amide I band at around 1650 cm-1 meant a decrease of total α-helix content. The decrease might be caused by the damage of considerable amounts of α-helix conformation that accounted for the major part of MPs. It should be pointed out that the peptide bonds in the α-helix conformation may form hydrogen bonds, and therefore, α-helix was very stable. In this study, the α-helix conformation largely despiralized after adding PFGS. The exposure of intramolecular hydrophobic groups appears to be conducive to formation of gel networks and appearance of new protein folding after despiralization. As a result, the enhancement of hydrophobic effects and gelation was promoted.
In the amide II region, there were N-H in-plane bending, C-N stretching, C-O in-plane bending and C-C stretching15, 16. With the addition of PFGS, the intensity of detection in the amide II region was also enhanced, possibly due to the presence of C-C, C-O and a large number of benzene rings in the Proanthocyanidins molecule. However, the strength in the amide II region still increased over time. The possible reason was that oxidation and dehydration expose the C-C expansion and C-O in-plane bending of the amide II region, thus detecting greater strength.
In the Raman spectrum, the C-H bond stretching vibration of aliphatic residues exists at 2500-3000 cm-117, 18. From Fig.10, it could be seen that the sample has a strong peak at 2930 cm-1, and compared with the blank contrast, the PFGS processing had a certain effect on the aliphatic C-H stretching vibration in the MP Raman spectrum19. With the gradual increase in the amount of PFGS, the more aliphatic residues are exposed after the protein was reversed, and more aliphatic residues appear, thereby promoting the hydrophobic interaction at the protein interface20, 21. The results show that as the concentration of PFGS increases, the gel has greater hydrophobic interaction and gelling properties. But the peak in the blank contrast detected on day 0 was larger than the PG group, because the blank contrast contained more free water and the binding water.
2.7. Rheological properties
Storage modulus (G’) and loss modulus (G’’) were important indicators of gel viscoelasticity. Dynamic rheological analysis was very useful for the study of protein functional properties during muscle processing, and it was helpful to study the gel formation process, which was the basis for the formation of good texture. The G’ represents the energy change caused by the change of elastic deformation in the gel structure, and the G’’ represents the change of viscosity during heating.
It could be seen from Fig.16-a and Fig.16-b that the G’ gradually decreases with the increase of temperature, reaching the lowest point at 48°C. Then continue to rise to 80℃, G’ continued to increase to a stable state, The study22, 23 also found similar results. The increase of G’ indicates the initial formation of gel or elastic protein network structure. The temporary decrease of G’ before 48℃ was due to the expansion and crosslinking of myosin heavy chain, possibly due to the denaturation of myosin tail, mainly involving the dissociation of non-covalent bonds and the temporary interaction between molecules, thereby increasing the mobility of myosin. After further heating up, the formation of hydrophobic groups and the interaction of disulfide bonds increase the interaction of protein aggregates and form a good silk structure, which leads to the increase of G’, indicating that the viscous sol state changes to the elastic gel network structure. Then, the G’ increased rapidly and reached a steady state at 72℃, which indicated that the gel network structure was completely formed.
When the PG concentration was higher than 0.05 g / kg, the G’ starting point and final point of PG were higher than those of the blank control group, indicating that the gel strength increased with the addition of PFGS. When the addition amount was 0.25 g / kg, it was found that there was a peak at 64.8 ℃, and then G’ continued to increase, indicating that substances with very weak strength were formed at 0.25 g / kg concentration. The samples after rheological completion were observed, which was consistent with the normal formation of gel in visual observation, and the morphology was flocculent condensation block.
With the addition of PFGS, G’’ also showed an upward trend, and G’ was more than five times that of G’’. With the increase of temperature, it reached the peak at about 70°C, and then showed a downward trend. When the addition amount was 0 - 0.20 g / kg, the viscosity of the gel increased with the increase of the addition amount and was higher than that of the blank contrast. However, when the addition amount was greater than or equal to 0.25 g / kg, the viscosity of PG was lower than that of the blank control.
The frequency scouldning in Fig.16-c and Fig.16-d showed that the average G’ and G’’ of PG increase with the addition of PFGS and scouldning frequency in the concentration range of 0 - 0.20 g / kg. However, it could be seen from 0.20 g / kg PG that the gel had shown certain instability, and the data were extremely low at 0.25 g / kg. At the same time, the data of G’ / G’’ could be obtained from Fig.17-f. When the addition amount was 0.05 g / kg, the composite viscosity data of PG and the blank contrast had no significouldt difference. When the addition amount was 0.05 - 0.20 g / kg, it could be seen that the addition of PFGS enhanced the strength of the gel, but there was also instability at 0.20 g / kg. With the addition of PFGS, the hardness and viscosity of the gel increased, but PG had instability at 0.20 g / kg, which was consistent with the detection of texture, Raman, and infrared.
It could be seen from Fig.17-e that with the increasing amount of Pas, the Persistence of CMPG continues to increase and collapses at 0.25g/kg, which was the same trend as the gel in rheology. Fig-g was the change trend of Storage modulus and Loss modulus with temperature and Pas addition. Comparing Fig.17-h, it could be seen that the change of Fig.17-g was not very stable. The error may be the temperature change of the heater. Caused by unevenness. The Hz change in Fig-h was a stable digital control, so the change trend was relatively stable. Fig.17-h and g could see that with the continuous increase of temperature, frequency and Pas addition, the storage modulus and Loss modulus of the gel were all increasing, but the growth rate of Storage modulus was significantly higher than that of Loss modulus, which proves that it was heating A gel was formed during the process, and it shows a certain degree of gel stability during frequency scanning
Fig.19-A was the 0th day of blank contrast, Fig.19-B was the 20th day of blank contrast, Fig.19-C was the 0th day of 0.20 g / kg PG, and Fig.19-D was the 20th day of 0.20 g / kg PG.
By comparing the microscopic images of the blank contrast and the original anthocyanin group at day 0, It was evident that the pure gel exhibited a rough surface with a disordered structure, implying that protein molecules were not fully unfolded and not welllinked to each other during gelation. With PFGS incorporation, the gel network became more regular and homogeneous with compact pores Fig.19-C. Overall, PFGS induced the development of a stable three-dimensional gel network. A highly interconnected and compact network structure might exhibit greater resistance to external stress and provide more space for entrapping water via capillary effects, thereby increasing gel strength and LF-NMR. In addition, based on the self-agglomeration effect of PFGS, the agglomerated PFGS could act as fillers to fill the network structure, and these filling effects seemed to be more pronounced at higher concentrations. In Fig.20, A, B, C was a solid figure with 0.20 and 0.25, 0.50 g / kg PFGS, respectively. It could be seen that with the increasing amount of PFGS, the morphology of the gel was broken, dried and irregular.
It could be seen that the surface and internal gel structure of the PG on day 0 was different, which was consistent with the problems found in LF-NMR, volume and texture detection of the block. Consideration may be caused by excessive contact between the epidermis of the outer layer of the gel and the solution during gel formation. So the PG removed all the surface gel on day 0. On the 20th day, it could be seen 20th day that a large area of collapse occurred in both groups of gels.
Carbonyl formation was commonly used as an indicator of the oxidative stability of proteins in biological systems 24, 25, and was often used as a marker of the oxidative stability in meat products26, 27. When the carbonyl group in the protein was formed, the function and structural properties of the protein will be impaired. Since there were many ways to form carbonyl groups, including amino acid side chain modification of especially proline, arginine, lysine, and threonine residues, and might lead to cross-linking or release of free carbonyls from the amino acid side chains24. Therefore, it was difficult to fundamentally inhibit the formation of carbonyl groups28. The only way to reduce the formation of carbonyl groups was to add antioxidant substances29, 30. Once the carbonyl group was formed, it could not be reduced, and other thiol-derived oxidation products might also be formed, which will further damage the protein.