Cold plasma-assisted protease hydrolysis modulates antigenicity, antioxidant properties, and bitterness of bovine casein

Figures (6) Tables (2)

Figure 1.
Effects of different treatments on (a) hydrolysis degree, (b) SDS-PAGE, (c) Tricine-SDS-PAGE, and (d) IgE binding capacity of CN. Lane M: molecular weight marker (10−180 kDa, 3.3−20.1 kDa); Lanes 1–6: ECN30, ECN30-CP, ECN60, ECN60-CP, ECN120, and ECN120-CP.
Figure 2.
Impact of various treatments on the structural characteristics of CN: (a) infrared spectroscopy; (b) secondary structure; (c) UV spectra; (d) fluorescence spectra; (e) surface hydrophobicity; (f) particle size distribution; (g) average particle size; and (h) potentials. Different letters indicate significant differences between groups (p < 0.05).
Figure 3.
Mass spectrometry analysis of hydrolysates of CNHs: (a) peptide chain mass distribution; (b) peptide chain length distribution; (c) bovine α_S2-casein (P02663); (d) bovine α_S1-casein (P02662); (e) bovine β-casein (P02666); (f) bovine κ-casein (P02668). The green bar height indicates the number of peptides, while the color intensity reflects the summed intensities of peptides overlapping this position.
Figure 4.
Results of in silico prediction on CN: (a) bioactive analysis of peptide sequences; (b) bitter peptide analysis.
Figure 5.
Effects of different treatments on the antioxidant activity of CN: (a) DPPH radical scavenging activity; (b) ABTS radical scavenging activity; (c) ferrous ion chelation activity; (d) total antioxidant capacity. Different letters indicate significant differences between groups (p < 0.05).
Figure 6.
Bitterness change of CN under different treatments: (a) total content of bitter free amino acids; (b) bitter taste value measured by electronic tongue. Different letters indicate significant differences between groups (p < 0.05).

Protein	Start	End	Intensity
Protein	Start	End	ECN60	ECN60-CP	ECN120	ECN120-CP
α_s1-CN	101	113	2.24 × 10⁸	1.45 × 10⁸	3.96 × 10⁷	0
α_s2-CN	132	143	2.24 × 10⁸	3.14 × 10⁸	1.48 × 10⁷	1.97 × 10⁷
	180	187	3.90 × 10⁸	3.39 × 10⁸	2.99 × 10⁷	0
	206	215	1.86 × 10⁹	9.20 × 10⁷	2.53 × 10⁷	0
β-CN	21	30	0	0	5.24 × 10⁷	4.35 × 10⁶
	60	66	1.00 × 10¹⁰	1.01 × 10¹⁰	1.39 × 10¹⁰	1.28 × 10¹⁰
	72	83	2.48 × 10¹⁰	2.00 × 10¹⁰	2.08 × 10¹⁰	1.51 × 10¹⁰
	124	133	1.03 × 10¹¹	7.84 × 10¹⁰	7.42 × 10¹⁰	5.96 × 10¹⁰
κ-CN	39	48	2.48 × 10⁹	1.24 × 10⁹	5.65 × 10⁸	2.19 × 10⁷
	81	99	0	4.33 × 10⁶	0	0
	118	122	1.18 × 10¹⁰	7.09 × 10⁹	3.27 × 10⁹	1.43 × 10⁹
	131	137	2.61 × 10⁸	3.59 × 10⁸	3.23 × 10⁹	6.71 × 10⁸

Table 1.

Changes in epitope intensity of CNHs.

Amino acid	CP	ECN60	ECN60-CP	ECN120	ECN120-CP
Asp	0.016 ± 0.004^ab	0.019 ± 0.003^ab	0.027 ± 0.005^b	0.007 ± 0.003^a	0.020 ± 0.013^b
Glu	0.037 ± 0.006^a	0.198 ± 0.006^b	0.240 ± 0.016^c	0.226 ± 0.039b^c	0.257 ± 0.017^c
Thr	0.020 ± 0.003^a	1.364 ± 0.034^bc	1.325 ± 0.010^b	1.435 ± 0.005^c	1.421 ± 0.082^c
Ser	0.026 ± 0.003^a	0.031 ± 0.002^a	0.023 ± 0.009^a	0.036 ± 0.009^a	0.036 ± 0.010^a
Gly	0.056 ± 0.007^a	0.128 ± 0.01^b	0.170 ± 0.019^c	0.132 ± 0.019^b	0.193 ± 0.024^c
Ala	0.012 ± 0.001^a	0.162 ± 0.019^c	0.145 ± 0.003^bc	0.114 ± 0.029^b	0.112 ± 0.031^b
Pro	0.080 ± 0.013^a	0.138 ± 0.013^b	0.141 ± 0.023^b	0.106 ± 0.002^a	0.135 ± 0.013^b
Lys	0.031 ± 0.011^a	0.071 ± 0.006^b	0.095 ± 0.004^c	0.096 ± 0.002^c	0.120 ± 0.007^d
Cys	0.422 ± 0.015^a	0.653 ± 0.016^b	0.775 ± 0.012^d	0.706 ± 0.023^c	0.865 ± 0.025^e
Val	0.091 ± 0.015^a	0.376 ± 0.017^b	0.445 ± 0.020^c	0.456 ± 0.009^c	0.536 ± 0.021^d
Met	0.060 ± 0.017^a	0.421 ± 0.032^d	0.354 ± 0.006^c	0.408 ± 0.009^d	0.315 ± 0.011^b
Ile	0.088 ± 0.013^b	0.042 ± 0.009^a	0.036 ± 0.006^a	0.073 ± 0.009^b	0.043 ± 0.006^a
Leu	0.053 ± 0.007^a	0.318 ± 0.032^c	0.266 ± 0.016^b	0.337 ± 0.011^c	0.323 ± 0.014^c
Tyr	0.075 ± 0.013^a	5.074 ± 0.060^c	4.744 ± 0.082^b	5.728 ± 0.088^d	5.072 ± 0.118^c
Phe	0.048 ± 0.015^a	7.135 ± 0.028^c	6.275 ± 0.006^b	7.944 ± 0.002^d	7.153 ± 0.021^c
His	0.021 ± 0.008^a	1.611 ± 0.024^c	1.228 ± 0.030^b	2.105 ± 0.028^d	1.635 ± 0.020^c
Arg	0.071 ± 0.012^a	0.557 ± 0.014^d	0.490 ± 0.014^c	0.456 ± 0.007^b	0.481 ± 0.005^c
Total	1.208 ± 0.027^a	18.300 ± 0.025^c	16.776 ± 0.036^b	20.365 ± 0.032^e	18.717 ± 0.023^d
Different letters indicate significant differences between groups (p < 0.05).

Table 2.

Effect of different treatments on the free amino acid composition of CN (mg/g); the last eight amino acids in the table belong to the category of bitter amino acids^[58].