Professor Yingyi Xu, Qiqihar University, et al.: Effects of heat treatment and transglutaminase on the properties of glycosylated oat protein gels

Among cereal foods, oat protein content ranked first (11.
19%~19.
85%)
.
Its protein composition of amino acids is balanced and reasonable proportion, of which globulin accounts for 70%~80% of the total protein content, mainly composed of 12S, 7S and 3S protein, due to the poor functional properties such as oat protein solubility, which limits its application
in the field of food processing.
It is a promising method
to modify proteins such as glycosylation and gelling and apply modified proteins as additives in food formulations.

In order to better apply modified protein as an additive in the food industry, Xu Yingyi and Ma Xinrui from the School of Food and Biological Engineering of Qiqihar University heat treated oat protein and its glycosylation products and TG modified it, respectively, determined the structure and gel properties of modified protein gel, and explored the effect
of glycosylation modification combined with heat treatment or TG modification on the gel properties of oat protein.
In order to provide a certain theoretical basis
for the development and application of oat protein.

1.
Process optimization analysis of HIGLGOP

As can be seen from Table 1, the influence of each factor on the strength of HIGLGOP gel is: protein fraction> pH value> reaction time > reaction temperature
.
From Table 2, it can be seen that protein mass fraction and pH value have significant effects on HIGLGOP gel strength (P<0.
05), while reaction time and reaction temperature have no significant effect<b11> on gel strength.
Combining Table 1 and Table 3, the optimal combination was obtained as A2B2C2D2, that is, protein mass fraction 8%, pH 10, reaction time 2 h, reaction temperature 95 °C, and the HIGLGOP gel strength was (186.
13±1.
97) g by the optimal combination, which was higher than the results of
the 9 orthogonal experiments in Table 1.
Subsequent experiments were performed
under this optimal combination of conditions.

2.
Process optimization analysis of LGOPIG

It can be seen from Table 4 that the influence of each factor on gel strength is: enzyme addition> protein fraction>pH value> reaction time > reaction temperature
.
It can be seen from Table 5 that the amount of enzyme added has a significant effect on the gel strength (P<0.
05), but the protein mass fraction, pH value, reaction time and reaction temperature have no significant effect on it.
Combining Table 4 and Table 6, the optimal combination was A4B3C1D2E2, that is, protein fraction 8%, enzyme addition amount 40 U/g, pH 6.
5, reaction time 2.
0 h, reaction temperature 55 °C, and the gel strength was (159.
64±1.
83) g by the optimal combination, which was higher than the results of<b11> the 16 orthogonal experiments in Table 4.
Subsequent experiments were performed
under optimal combinations.

3.
Analysis of oat protein gel texture properties

It can be seen from Figure 1 that compared with HIGOP, the elasticity and hardness of the other three oat protein gels are significantly improved (P<0.
05), and among the four oat protein gels, LGOPIG has the best<b10> texture properties.
Compared with HIGOP, the elasticity, hardness and adhesiveness of LGOPIG are increased by 7.
27%, 9.
49% and 13.
01%,
respectively.
The elasticity and adhesiveness of LGOPIG were significantly higher than those of HIGLGOP (P<0.
05), and the texture properties of LGOPIG were significantly higher than that of OPIG(P<0.
05).
<b12>

4.
Analysis of oat protein gel water holding capacity

It can be seen from Figure 2 that among the four oat protein gels, LGOPIG has the best
water holding capacity.
Compared with HIGOP, the water holding capacity of the other three oat protein gels was significantly improved (P<0.
05), and the water holding capacity of HIGLGOP, OPIG and LGOPIG was increased by 14.
21%, 4.
86% and 22.
22%,<b11> respectively 。 The water holding capacity of the two enzyme-induced protein gels (OPIG and LGOPIG) was higher than that of the two heat-induced protein gels (HIGOP, HIGLGOP), respectively.
The water holding capacity of the two glycosylated modified protein gels (HIGLGOP and LGOPIG) was higher than that of the two unglycosylated modified protein gels (HIGOP and OPIG),
respectively.

5.
Analysis of hydrophobicity on the surface of oat protein gel

It can be seen from Figure 3 that the H0 of the two enzyme-induced protein gels (OPIG, LGOPIG) is higher than that of the two heat-induced protein gels (HIGOP, HIGLGOP), respectively.
The H0 of the two glycosylated modified protein gels (HIGLGOP and LGOPIG) was lower than that of the two unglycosylated modified protein gels (HIGOP and OPIG),
respectively.
It is possible that H0 is determined by the degree of exposure of surface residues of the protein
.
The more hydrophobic residues exposed to the molecular surface, the stronger
the H0.
After oat protein was catalyzed by TG, the protein structure changed, the polypeptide chain unfolded, and the hydrophobic region was exposed from the internal molecular group, which increased H0, which was consistent with
the conclusion of Zang Xueli et al.

6.
SDS-PAGE analysis of oat protein gel

It can be seen from Figure 4 that compared with heat-induced protein gels (HIGOP, HIGLGOP), TG crosslinked protein gels (OPIG, LGOPIG) have a large number of macromolecular weight protein crosslinking products at the top of the separation gel, and the colors at 31.
0 and 21.
0 kDa are significantly lighter than heat-induced protein gels, and at the same time, there are large molecular weight protein accumulations in the tank that cannot migrate down and cannot enter the gel (at the arrow).

The results showed that TG caused intermolecular or intramolecular crosslinking of oat proteins to form macromolecular mass crosslinking products
.

7.
Fluorescence spectroscopy analysis of oat protein gel

It can be seen from Figure 5 that the fluorescence intensity of the two enzyme-induced protein gels (OPIG and LGOPIG) is higher than that of the two heat-induced protein gels (HIGOP and HIGLGOP), respectively.
The fluorescence intensity of the two glycosylated modified protein gels (HIGLGOP and LGOPIG) was higher than that of the two unglycosylated modified protein gels (HIGOP and OPIG),
respectively.
The maximum fluorescence emission wavelengths of HIGOP, HIGLGOP, OPIG and LGOPIG were 358, 355, 349 and 353 nm, respectively, indicating that the maximum fluorescence emission wavelength of the protein after TG crosslinking decreased and the blue shift
occurred.
The fluorescence intensity of enzyme-induced protein gels is higher than that of heat-induced gels, because TG crosslinks oat proteins, causing changes in protein structure, exposure of chromogenic amino acids such as tryptophan and tyrosine, and increased fluorescence intensity
.

8.
FTIR analysis of oat protein gel

As shown in Figure 6, compared with unglycosylated modified oat protein gels (HIGOP, OPIG), the absorption peaks of glycosylated protein gels (HIGLGOP, LGOPIG) widened in the range of 3 200~3 700 ^cm-1, which proved that after covalent binding of oat protein and lactose, the number of hydroxyl groups of the complex increased, causing the expansion vibration
of C—OH.
In the amide I band region (1 600~1 700 ^cm-1), absorption leads to tensile vibration of C=O bonds, so it is considered to be the most sensitive region
for studying protein structural changes.
It can be seen from Figure 6 that the waveforms of the TG-modified proteins are shifted to the high wavenumber by about 1~2 ^cm-1
.
This shows that under the influence of TG, the protein structure is unstable and the hydrogen bond action is weakened, so that the bond length of the C=O chemical bond decreases, while the telescopic vibration of the chemical bond is inversely proportional to the square root of the bond length, and the expansion vibration frequency increases due to the decrease of bond length, thus leading to an increase
in wavenumber.

9.
Microstructure analysis of oat protein gel

As can be seen from Figure 7, the surface structure of LGOP gels (Figures 7B and D) becomes more loose and porous
compared to unglycosylated oat protein gels (Figures 7A and C).
This may be that the glycosylation reaction forms an inhomogeneous structure that changes the protein structure, resulting in a change
in the functional properties of the oat protein gel.
Compared to oat proteins crosslinked without TG (Figures 7A and B), protein size in TG crosslinked oat protein gels (Figures 7C and D) changes greatly and pore size decreases in
gel space.
This shows that TG can reduce disordered aggregation between protein molecules to form a more ordered structure
.

Conclusion