Professor Li Xin of Nanchang University, et al.: Effect of ozone treatment on the structure and allergenicity of bovine whey protein

Whey protein accounts for about 20% of the total protein content of bovine milk, which is usually composed of globular proteins such as α-lactalbumin, β-lactoglobulin, bovine serum albumin, lactoferrin and immunoglobulin (Ig).

Among them, β-lactoglobulin and α-lactalbumin have the highest content, accounting for 52% and 19%
of whey protein content, respectively.
Whey protein has high nutritional value, studies have shown that whey protein can improve people's sleep quality, promote the development of gastrointestinal function in infants and young children, and enhance resistance
.
However, β-lactoglobulin and α-lactalbumin in whey are the main allergens
in bovine milk.

Ozone has a strong broad-spectrum bactericidal effect and is an efficient and safe "green disinfectant"
.
Xiong Ziyi and Li Xin* from the State Key Laboratory of Food Science and Technology of Nanchang University took bovine whey protein as the research object to explore the changes of amino acid composition, spatial structure, sulfhydryl group and disulfide bond content of whey protein before and after ozone treatment, and then deeply understood the effect of
ozone treatment on the structural characteristics of whey protein.
In addition, the indirect competitive enzyme-linked immunosorbent assay (ELISA) was used to detect the change of whey protein sensitization after ozone treatment, in order to explore new methods
for ozone to improve the food safety of milk protein and improve protein function.

1.
Electrophoretic analysis of ozone-treated whey protein

As shown in Figure 2, the main proteins of WPC are α-lactalbumin with a molecular mass of 14.
2 kDa and β-lactoglobulin
with a molecular mass of 18.
4 kDa 。 In lane 2 (ozone treatment 5 s) and lane 3 (ozone treatment 10 s), the main components of whey protein did not change significantly, but the color of the protein band with a molecular mass of about 34 kDa became darker, which may be due to the oxidation of ozone, so that the disulfide bond is formed between free cysteine in the adjacent β-lactoglobulin monomer, resulting in more β-lactoglobulin dimers, and the free cysteine of β-lactoglobulin is located in the center of the protein molecular structure, and the disulfide bond formed is not easily reduced by β-mercaptoethanol
。 With the further extension of the ozone treatment time, compared with the untreated samples, the concentrations of α-lactalbumin and β-lactalbumin monomers in the whey protein after ozone treatment decreased, and the polymer mass bands were more obvious, indicating that ozone treatment caused the formation of different degrees of polymers
between whey protein monomers.

2.
Effect of ozone treatment on the primary structure of whey protein

Effect of ozone treatment on amino acid content of whey protein

It can be seen from Table 2 that the relative contents of serine, glycine, valine, methionine, isoleucine, tyrosine, histidine and arginine in the ozone treatment group were lower than those of the samples without ozone treatment, and the changes of glycine, methionine, tyrosine and histidine decreased significantly with the extension of ozone treatment time, while the relative content of other amino acid components did not decrease
significantly.

Effect of ozone treatment on whey protein sulfhydryl and disulfide bonds

As shown in Figure 3, under the ozone treatment conditions of 5, 10, 15, 20 s and 25 s, the free sulfhydryl content of the untreated sample and the ozone treated sample was 5.
78, 4.
49, 3.
85, 3.
01, 2.
92 μmol/g and 2.
13 μmol/g, respectively, the total sulfhydryl content was 14.
98, 14.
73, 14.
12, 13.
80, 13.
60 μmol/g and 12.
97 μmol/g, while the disulfide bond content was 4.
60 and 12.
97 μmol/g, respectively.
5.
12, 5.
13, 5.
39, 5.
34 μmol/g, and 5.
42 μmol/g
.
Compared with the untreated sample, the ozone treated sample had a slight decrease in both free sulfhydryl and total sulfhydryl content, while the disulfide bond content was slightly increased
.
The main reason for the decrease in sulfhydryl content may be that the oxidation of sulfur-containing amino acids produces different residues, such as cysteine, which is sensitive to oxidants, oxygen radical action sites are very shallow, oxidation may form sulfinic acid/sulfonic acid, and methionine is oxidized to methionine sulfone
.
The increase in disulfide bond content is due to the aggregation of sulfhydryl groups on the side chain of two molecules of cysteine to form new disulfide bonds
.

3.
Effect of ozone treatment on the secondary structure of whey protein

As shown in Figures 4 and 3, ozone treatment converts the secondary structures in whey protein to each other compared to untreated whey protein
.
With the extension of time, the relative content of α-spiral decreased, the relative content of β-folding increased slightly, the overall relative content of β-angle changed less, and the relative content of random curl showed a trend
of first increasing and then decreasing.
This may be due to the short-term ozone treatment that causes the whey protein to unfold and the protein structure to partially unfold, so that its well-ordered secondary structure tends to be loose, and the natural spherical form is lost, making it more elastic
.
With the extension of the processing time, the protein molecules aggregate and the content of disulfide bonds increases, and the presence of disulfide bonds helps to strengthen and stabilize the protein structure, making the molecular structure more orderly
.

4.
Effect of ozone treatment on the spatial structure of whey protein

Ozone-treated whey protein UV spectroscopy

As can be seen from Figure 5, the UV spectrum of WPC has a characteristic absorption peak around 280 nm, which is mainly caused
by the aromatic heterocyclic π→π* transition of tryptophan and tyrosine residues on the protein peptide chain.
When the ozone treatment time was 5 s, the characteristic absorption peak of the sample did not shift, but the intensity was higher, indicating that short-term ozone treatment would not change the sidechain group distribution of tryptophan and tyrosine residues, but would expand the spatial structure of whey protein, expose more tryptophan and tyrosine residues, and loosen the tertiary structure
。 With the extension of ozone treatment time, the maximum absorption peak intensity of whey protein gradually increased, and there was a more obvious blue shift, which indicated that the spatial structure of protein and amino acid side chain groups would change significantly after prolonged ozone treatment, resulting in changes
in the microenvironment of amino acids with ultraviolet absorption capacity.

Ozone-treated whey protein endogenous fluorescence spectroscopy

As shown in Figure 6, when the ozone treatment time was 5 s, the endogenous fluorescence of whey protein did not change significantly, but with the extension of the treatment time, the endogenous fluorescence of whey protein gradually decreased, and when the treatment time was 25 s, its characteristic peak was only 6.
94% of the untreated sample, and a blue shift
of 9 nm occurred.
This may be due to the fact that tryptophan has a low single-electron oxidation potential energy and is easily converted into unstable tryptophan radicals, which in turn are oxidized by ozone to kynurenine, which reduces
the endogenous fluorescence intensity.
As the reaction progresses, the aggregation of proteins causes the unoxidized tryptophan in the protein molecule to return to the non-polar environment, and its maximum absorption wavelength is blueshifted
.

3D fluorescence spectroscopy analysis of ozone-treated whey protein

As can be seen from Figure 7 and Table 4, ozone treatment significantly reduces
the peak intensity of peak A and peak B.
Comprehensive analysis shows that ozone treatment converts some tyrosine and tryptophan residues in whey protein into derivatives, resulting in fluorescence quenching, while the aggregation of protein molecules reduces the exposure of hydrophobic amino acids such as tryptophan and changes
the tertiary structure of proteins.

Ozone-treated whey protein surface hydrophobicity analysis

This is shown
in Figure 8.
Under the condition of ozone treatment for a short time (5, 10 s), the surface hydrophobicity of whey protein showed an increasing trend, but with the extension of treatment time (15~25 s), the surface hydrophobicity of the sample decreased significantly, and it was significantly negatively correlated
with the treatment time.
This change can be explained
by a change in protein conformation during oxidation.
When the ozone treatment time is short, ozone changes the spatial structure of whey protein, its structure becomes looser, and the hydrophobic amino acids buried in the protein in the natural state are exposed to the protein surface, making its surface hydrophobicity enhanced
.
However, with the extension of ozone time, more and more protein monomers have covalent or non-covalent binding between each other, resulting in different degrees of polymers, and the hydrophobic groups on their surface are buried inside the multimers, resulting in a significant decrease
in the hydrophobicity of protein surfaces.
In addition, the oxidation of ozone leads to changes in the content of hydrophobic amino acids such as methionine, which is also a key factor in
the change of hydrophobicity of protein surfaces.

5.
Changes in the ability of ozone to treat whey protein to bind to IgG and IgE