Reduced antigenicity of β-lactoglobulin by conjugation with glucose through controlled Maillard reaction conditions

Figures & data

Table 1. Coded and uncoded settings of the independent variables for dry-heating according to a central composite rotatable design.

	Independent variables
Coded level	WR	Temperature (°C)	Time (h)
1.682	7.83	60	120
1	6.28	56	100.5
0	4	50	72
−1	1.72	44	43.5
−1.682	0.17	40	24
Note: WR, weight ratio of WPI to glucose.

Table 2. Changes in content of free amino groups and brown colour during the dry-heating of WPI and glucose.

Samples	Content of free NH2 groups (%)^a	OD420 ^a
Native WPI	100±0.54	0.017±0.001
Dry-heating WPI (40°C, 120 h)	97.5±0.27	0.018±0.001
Dry-heating WPI (50°C, 120 h)	94.8±0.53	0.019±0.001
Dry-heating WPI (60°C, 120 h)	88.7±0.64	0.021±0.001
WPI–G (WR 7.83:1, 50°C, 72 h)	48.9±0.36	0.047±0.001
WPI–G (WR 4:1, 50°C, 72 h)	34.1±0.62	0.056±0.001
WPI–G (WR 0.17:1, 50°C, 72 h)	33.6±0.27	0.087±0.002
WPI–G (WR 4:1, 40°C, 72 h)	56.0±0.44	0.025±0.001
WPI–G (WR 4:1, 50°C, 72 h)	34.1±0.62	0.056±0.001
WPI–G (WR 4:1, 60°C, 72 h)	20.7±0.54	0.068±0.003
WPI–G (WR 4:1, 50°C, 24 h)	50.7±0.44	0.031±0.001
WPI–G (WR 4:1, 50°C, 72 h)	34.1±0.62	0.056±0.001
WPI–G (WR 4:1, 50°C, 120 h)	24.0±0.20	0.098±0.002
^aMeans±SD (standard deviation) for three experiments. Note: WR, weight ratio of WPI to glucose.

Table 3. Full factorial central composite design matrix for the antigenicity of β-LG in WPI–glucose conjugates.

	Independent variables^a			Dependent variable
Assay	WR	Temperature (°C)	Time (h)	β-LG antigenicity (µg/ml)^b	Inhibition rate (%)
1	1.72 (−1)	44 (−1)	43.5 (−1)	59.76±2.01	77.86
2	1.72 (−1)	44 (−1)	100.5 (1)	22.21±0.96	91.77
3	1.72 (−1)	56 (1)	43.5 (−1)	11.41±0.30	95.77
4	1.72 (−1)	56 (1)	100.5 (1)	21.2±1.31	92.15
5	6.28 (1)	44 (−1)	43.5 (−1)	154.94±4.29	42.59
6	6.28 (1)	44 (−1)	100.5 (1)	88.95±0.71	67.04
7	6.28 (1)	56 (1)	43.5 (−1)	47.86±2.45	82.27
8	6.28 (1)	56 (1)	100.5 (1)	109.02±1.77	59.61
9	0.17 (−1.682)	50 (0)	72 (0)	10.7±0.23	96.04
10	7.83 (1.682)	50 (0)	72 (0)	100.31±3.48	62.83
11	4 (0)	40 (−1.682)	72 (0)	106.04±2.6	60.71
12	4 (0)	60 (1.682)	72 (0)	12.63±1.48	95.32
13	4 (0)	50 (0)	24 (−1.682)	90.61±2.00	66.43
14	4 (0)	50 (0)	120 (1.682)	19.33±0.29	92.84
15	4 (0)	50 (0)	72 (0)	21.8±1.52	91.92
16	4 (0)	50 (0)	72 (0)	17.79±1.17	93.41
17	4 (0)	50 (0)	72 (0)	22.42±1.01	91.69
18	4 (0)	50 (0)	72 (0)	19.88±0.67	92.63
19	4 (0)	50 (0)	72 (0)	18.46±0.96	93.16
20	4 (0)	50 (0)	72 (0)	20.63±1.58	92.36
21	4 (0)	50 (0)	72 (0)	23.05±1.13	91.46
22	4 (0)	50 (0)	72 (0)	19.51±1.42	92.77
23	4 (0)	50 (0)	72 (0)	20.25±1.08	92.50
^aValues in parentheses are the coded levels of independence variables. ^bMeans±SD (standard deviation) for three experiments.

Table 4. Regression coefficients for the linear regression model for prediction of the antigenicity of β-LG.

	Full model		Fitted model
	b-coefficient	P	b-coefficient	P
Intercept	20.30		20.30
Linear
Weight ratio	31.99	0	31.99	0
Temperature	−21.49	0	−21.49	0
Time	−11.16	0.011	−11.16	0.009
Quadratic
Weight ratio^2	13.57	0.002	13.57	0.001
Temperature^2	14.92	0.001	14.92	0.001
Time^2	13.38	0.002	13.38	0.001
Interactions
Weight ratio×temperature	−4.71	0.359	–	–
Weight ratio×time	2.87	0.573	–	–
Temperature×time	21.81	0.001	21.81
Other statistics
R ^2	0.886		0.892
F	19.94	0.0001	26.83	0.0001

Figure 1.  3D response surface plot and 2D contour plot using the b coefficients from the fitted model (Table 4) for the effect of weight ratio of WPI to glucose (X ₁) and temperature (X ₂) on the antigenicity of β-LG (Y) at the reaction time for 72 h.

Figure 2.  3D response surface plot and 2D contour plot using the b coefficients from the fitted model (Table 4) for the effect of weight ratio of WPI to glucose (X ₁) and time (X ₃) on the antigenicity of β-LG (Y) at a temperature of 50°C.

Figure 3.  3D response surface plot and 2D contour plot using the b coefficients from the fitted model (Table 4) for the effect of temperature (X ₂) and time (X ₃) on the antigenicity of β-LG (Y) at weight ratio 4:1 of WPI to glucose.