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Highlights
- The interaction between fish gelatin (FG) and silver fungus polysaccharide (AP) was analyzed
- At turbidity with a mass ratio of 1:1 and a pH of 4.
0, the maximum complexation reaction occurs - The formation of composite agglomerates is mainly driven by static electricity
- FG-AP composite agglomerates show a porous network structure
- FG-AP composite agglomerates have high apparent viscosity
Gelatin, one of the basic materials of protein-polysaccharide complex aggregates, is a hydrolysate of collagen that can be extracted
from mammals and fish.
It is well known that mammalian gelatin has its limitations in terms of new diseases, such as prion disease and foot-and-mouth disease, which do not meet the needs of
Muslims and Jews.
Therefore, fish gelatin (FG) has shown advantages
as an alternative to mammalian gelatin.
However, due to its low content of hydroxyproline and proline, it functions poorly
in terms of gelatinization, rheology, and stability.
To overcome these problems, some studies have proposed the idea that
fish gelatin can be modified by enzyme (MTGase, tyrosinase, and laccase), chemical (phosphorylation, aldehydes, phenolic reactions), and physical (electrolyte or non-electrolyte substances and mechanical processing) methods.
Among them, mixing polysaccharides with fish gelatin is considered a promising method
.
Protein-polysaccharide complexes have many applications, such as fat replacement, emulsions, and bioactive encapsulation
.
In general, the formation of protein-polysaccharide complexes can be monitored by turbidity titration, which may be affected
by pH, protein/polysaccharide ratio, total biopolymer concentration, and ionic strength.
Protein-polysaccharide complexes can have an impact
on their physicochemical properties.
People pay more attention to the rheological properties of fish gelatin-polysaccharide complexes, especially viscosity, which is one of
the most important commercial indicators of gelatin.
Studies have mentioned that by adding natural mucus, such as xanthan gum, gum arabic and kappa-carrageenan, the viscosity
of fish gelatin can be significantly improved.
The polysaccharides obtained from Tremella polysaccharides (AP) are named Tremella polysaccharides, which have many physical properties and physiological functions
in moisturizing, gelatinization, immunity, anti-aging, antioxidant, hypoglycemia, hypolipidemia, etc.
Recently, several works have reported new complex agglomerates
based on AP and other proteins, including whey protein isolate and zelamin.
However, there are few reports of
interaction behavior of FG-AP mixtures.
In addition, with the change of protein-polysaccharide complex condensation system, its mechanism is also different
.
Therefore, it is necessary to study the interaction mechanism
between FG and AP.
Feng Jiawen, Wang Shaoyun*, Shi Xiaodan* of Fuzhou University and others evaluated the possibility of interaction between FG and AP by turbidimetry, including pH, protein-polysaccharide ratio, total biopolymer concentration, and the influence of
additive type and concentration.
In addition, several analytical techniques have been used to characterize the structure and physicochemical properties of FG-AP condensates, including fluorescence measurement, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), and rheological testing
.
Results and DiscussionFactors
Affecting FG-AP Interactions in Aqueous SolutionsInfluence of pH plays an important role
in the formation of protein and polysaccharide complexes.
The charge balance is mainly due to the pH of the environmental conditions, which further contributes to the strength
of the electrostatic action.
In Figure 1A, B, the determination
of turbidity and zeta potential from pH 2.
0 to pH 8.
0 is observed.
As a control, the turbidity of the pure FG solution remained a constant low value
at all pH conditions.
However, due to self-aggregation, AP shows a small peak
at pH 4.
0.
In addition, the absorbance values of the FG-AP complex are consistently higher than those of FG and AP and are essentially pH dependent
.
Based on previous reports, four critical pH points (pHc, pHφ1, pHopt, and pHφ2)
were identified.
As shown in Figure 1B, the pI of FG is observed around pH 7.
0, while AP is always negatively charged
.
At pH > pHc (7.
0), the turbidity of the FG-AP complex remains stable
.
This phenomenon suggests that the interaction between FG and AP may be weak, which is related to
their negative charge.
When the pH is equal to pHc (7.
0), soluble complexes are created
due to their opposite charges.
When the pH drops to pHφ1 (6.
0), the turbidity increases
significantly with the appearance of insoluble complexes.
At pH 4.
0 (expressed as pHopt), turbidity reaches its maximum due to the presence
of a large number of insoluble complexes.
This trend can be attributed to the neutralizing charge
in the FG-AP complex.
Thereafter, as the pH decreases, the turbidity drops sharply, indicating that the insoluble complex begins to dissociate
.
When the pH <φ2 (3.
0), the turbidity value is low and constant<b137> due to the termination of electrostatic attraction.
Another key factor affecting the formation of compounds by the influence of biopolymer ratio is the FG-AP ratio
.
According to Figure 1C, regardless of how the protein content decreases or increases, the maximum turbidity conducive to the formation of the FG-AP complex is a ratio of 1:1
for FG and AP.
This result shows that each polysaccharide chain provides enough binding sites for each protein to bind
to each other.
In addition, increasing the proportion of AP shifts the turbidity curve peak towards a higher pH, while increasing the proportion of FG shifts the curve peak towards a lower pH due to the presence of more positive charges
on the surface of FG.
When the FG-AP complex is excessive, it has more positive charge, which means that the pH must move to a higher value to attract the negative charge
of the AP.
The effect of the total biopolymer concentration changes in the total biopolymer concentration also affect the process of the
complex.
Different concentrations of FG-AP complexes produce similar turbidity curves, with a maximum turbidity of pH 4.
0
in Figure 1D.
However, when the concentration of biopolymers is too low, the critical pH value is difficult to distinguish
.
As the concentration increases, the turbidity tends to increase, which can be interpreted by the fact that insoluble complexes have a good chance of formation
at high concentrations.
This result is consistent with previous discussions that the total concentration of biopolymers is an important influencing factor
.
However, contrary observations have been found in other studies, with reports of turbidity values peaking
at a total concentration of 0.
75%.
The argument in favor of this phenomenon is that the release of counterions screens the charge, which can lead to an increase
in solubility.
The effect of additive type and concentration was studied by adding unstable agents such as salt, urea, and sodium lauryl sulfate (SDS), and the interaction forces
of the FG-AP complex.
All the turbidity in Figure 1E showed a decreasing trend with the increase of the additive concentration, and the turbidity decreased sharply after the addition of SDS
.
FG-AP complexes containing divalent ions decreased more than monovalent ions, while complexes containing urea decreased more slowly
.
Salt ions, SDS and urea can act as electrostatic shielding, electrostatic destruction and hydrophobic interactions, and interfere with hydrogen bonds
, respectively.
Therefore, electrostatic, hydrophobic interactions, and hydrogen bonding can play the role of inducing the formation of complexes between FG and AP, where electrostatic interaction is the main force
.
Fluorescence spectroscopy Figure 2 shows the fluorescence spectra
of FG and FG-AP complexes at pH 4.
0 and 7.
0.
The interaction of proteins with polysaccharides is studied by fluorescence spectroscopy, which detects changes in
the intrinsic fluorescence of proteins.
In general, the two effective intrinsic fluorophores in fish gelatin are tyrosine (Tyr) and phenylalanine (Phe).
However, Phe's photoluminescence quantum yield is low, so Tyr is the main force of
fluorescence.
When AP is added to FG, fluorescence intensity shows a decrease accompanied by redshift, which means that the specificity of the local microenvironment becomes slightly more hydrophilic and the FG-AP complex can be successfully formed
by electrostatic interaction.
In the above study, spectroscopic techniques were used to illustrate the phase behavior and interaction mechanism
of FG and AP in aqueous solutions.
The results showed that their strongest complexation occurred at pH 4.
0, so the complex azeotropes were freeze-dried and collected
.
In addition, FG-AP complexes
formed at pH 3.
5, pH 3.
0 and pH 7.
0 were collected.
Four samples were used for SEM and rotational rheometer testing
.
The microstructure characterization of FG-AP condensates with different pH values was characterized by SEM
.
As shown in Figure 3, the shape of the FG-AP agglomerate is porous at pH 3.
0, 3.
5, and 4.
0, and at pH 7, it appears as a sheet structure
。 In addition, the agglomerates formed at pH 3.
5 are more compact
than at pH 3 and 4.
The mesh structure is the result of
electrostatic attraction between FG and AP.
The stronger the interaction between them, the denser
the network structure formed.
Rheological Properties Figure 4A shows the viscosity of FG-AP aggregates at different pH values, indicating that the viscosity of all agglomerates decreases with shear rates from 0.
1 to 1001 s-1, indicating typical shear-thinning flow behavior
.
FG-AP agglomerates can be found to have the greatest viscosity at pH 3.
5
.
As previously reported, the strength of the electrostatic interaction between protein and polysaccharide is highly correlated
with its viscosity.
Thus, FG-AP aggregates at pH 3.
5, i.
e.
electrically neutral, can form a tighter structure
.
In addition, the viscoelastic properties
at different pH values are observed in Figure 4B.
First, strain scanning experiments were performed in the linear viscoelastic region (LVR) to set the strain to 0.
1%.
With the exception of FG-AP aggregates at pH 7.
0, the storage modulus (G′) and loss modulus (G′′) of the other samples predominate, have no intersection, and exhibit elastogel-like behavior
.
Among FG-AP aggregates at pH 3.
5, G′ and G" reach the highest values, which is attributed to the compact network structure
.
This result is consistent
with the results of viscosity and microstructure.
According to the above data, the strongest interaction between FG and AP occurs at pH 3.
5
at a certain concentration of FG and AP.
Further analysis compares the structural and thermal properties
of FG-AP agglomerates formed at pH 3.
5.
Structural and thermal properties of FG-AP condensates formed at pH 3.
5 FTIR analysis uses FTIR spectroscopy to characterize the functional and molecular structure
of the sample.
In Figure 5A, the characteristic peaks of FG appear at 3290 cm-1 (amide A, O-H and N-H stretching), 2932 cm-1 (amide B, CH2 asymmetric stretching), 1634 cm-1 (amide I, C=O stretching), 1530 cm-1 (amide II, N-H bending and C-N stretching), and 1235 cm-1 (amide III, C-N tensile, N-H bending and CH2 rocking vibration).
In the AP spectrum, peaks around 3335 and 2921 cm-1 are related
to tensile vibration bands of O-H and C-H, respectively.
Meanwhile, the absorption peaks of 1602 and 1412 cm-1 include asymmetric and symmetrical tensile vibrations
of COO-.
In addition, vibrations from C-O-C or C-O-H groups in the frequency band 1025 cm-1
.
These spikes indicate the presence of uronic acid
in the AP.
In particular, 800 cm-1 corresponds to mannose residues
.
Compared to AP alone, it can be seen that the peak of the FG-AP condensate at 3292 cm-1 shifts to the low-wavenumber side, i.
e.
, from 3335 cm-1 to 3292 cm-1.
However, compared to FG, its peak is close to that of FG-AP.
This may be due to the formation of hydrogen bonds between FG and AP, or the protein may make up a relatively high proportion
of FG-AP condensates.
In addition, the disappearance of peaks 1235, 1602, and 1412 cm-1 of FG-AP condensate confirms electrostatic interactions
with gelatin amino groups and polysaccharide carboxyl groups.
X-ray diffraction analysis detected the crystalline structure and size in FG, AP, and FG-AP condensates by X-ray diffraction, and the results are shown in
Figure 5B.
The X-ray diffraction pattern of all samples shows broad diffraction peaks, indicating that they are amorphous polymers
.
The diffraction peak of the AP is located at 8.
78°
.
The two diffraction peaks of FG are at 6.
76° and 20.
51°
.
FG-AP condensate has two wide peaks at 9.
04° and 19.
81°, similar to those shown in FG, which is related to
the triple helix structure of gelatin.
In addition, the X-ray diffraction peak of FG-AP condensate is higher in intensity than FG, supporting the successful formation of complexes
between FG and AP.
DSC Analysis Figure 5C shows DSC curves
for FG, AP, and FG-AP aggregates.
There are no endothermic peaks in AP, which indicates that the thermal stability of the polysaccharide is very good
.
In contrast, endothermic peaks of FG and FG-AP condensates were observed
at 81.
6 °C and 78.
9 °C, respectively.
The denaturing temperature value decreases with the addition of AP, demonstrating the electrostatic interaction
between FG and AP.
Corresponding author profile: Professor Wang Shaoyun
Executive Dean, College of Biological Science and Engineering, Fuzhou University
Dean of the Institute of Marine Science and Technology, Fuzhou University
Shaoyun Wang, Ph.
D.
, second-level professor, doctoral supervisor, executive dean of the School of Biological Science and Engineering of Fuzhou University, postdoctoral fellow at the University of Wisconsin (UW-Madison) and UC-Davis, was selected as a leading talent in scientific and technological innovation of the National "10,000 Talents Program", a leading talent in scientific and technological innovation for young and middle-aged people of the Ministry of Science and Technology, a provincial A high-level talent, a provincial high-level innovation talent, and a provincial scientific and technological innovation leader
。 He is also a director of the Chinese Society of Food Science and Technology, the vice chairman of the Fujian Health Engineering Society, the vice chairman of the Fujian Food Science and Technology Society, the editorial board member of Food Science and Human Wellness, Journal of Future Foods, Hans Journal of Food and Nutrition Science, Food Science, Food Industry Science and Technology, and Food Science of Scientific editor-in-chief of Animal Products, one of the first translation experts
of "Chinese and Foreign Food Technology".
He has presided over more than 30 projects at the provincial and ministerial level, compiled 8 books, authorized 69 invention patents, and published 300 academic papers, including 230 in SCI/EI
.
The achievements he presided over won the International ICOFF Academic Conference Award, the first prize of China's industry-university-research cooperation innovation achievements, the first prize of National Food Industry-University-Research Excellent Scientific Research Achievements, the first prize of Science and Technology Progress Award of China Federation of Chemical Industry, the first prize of Provincial Science and Technology Progress Award, the second prize of Provincial Science and Technology Progress Award, and the second prize of Provincial Natural Science Award
.
He has won the Baosteel Outstanding Teacher Award, the Provincial Excellent Teacher Award, the Provincial Outstanding Scientific and Technological Worker Award, the Lu Jiaxi Outstanding Mentor Award and the Famous Teacher Award
.
He was invited to serve as the "Changjiang Scholar" Distinguished Professor of the Ministry of Education and the review expert
of the National Natural Science Foundation of China.
Interaction between fish gelatin and tremella polysaccharides from aqueous solutions to complex coacervates: structure and rheological properties
Jiawen Feng, Han Tian, Xu Chen, Xixi Cai, Xiaodan Shi*, Shaoyun Wang*
Institute of Food and Marine Bio-Resources, College of Biological Science and Engineering, Fuzhou University, Fuzhou, 350108, China
*Corresponding authors.
Abstract
In the food industry, there has been a debate about the interactions between fish gelatin and polysaccharide with the interaction mechanism in different solute and solvent systems still unclear.
This article tries to explore interactions between fish gelatin (FG) and Tremella polysaccharides (AP) by the turbidity titration.
Especially, the turbidity was greatly influenced by pH, polymer ratio, total polymer concentration, and ionic strength.
Optimally, the most complex coacervates could be electrostatically formed at a mass ratio of 1:1 and pH 4.
0, which was collected for subsequent characterization.
In addition, there was a positive correlation between the turbidity and the total concentration, while the ionic strength was just reverse.
Modern instruments including FTIR, XRD, DSC, SEM and rotational rheometer were adopted to characterize the complex coacervates formed at different pH.
Results showed that the FG–AP complex coacervates showed porous network structure and had the strongest apparent viscosity with viscoelastic modulus at pH 3.
5.
These results suggested important implications in how we make a good alternative for fat in food dairy.
