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Effects of pH, temperature, enzyme-to-substrate ratio and reaction time on the antigenicity of casein hydrolysates prepared by papain

Xiaoyu Liu, Yongkang Luo* and Zheng Li

Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China

(Received 29 March 2011; final version received 28 June 2011)

The effects of pH, temperature, enzyme-to-substrate ratio and reaction time on the antigenicity of casein hydrolysates were investigated. Response surface methodology (RSM) was employed to optimise the reaction conditions. Enzy- matic hydrolysis with papain could reduce the antigenicity of a-casein and b-casein effectively and the reduction of antigenicity could be controlled by regulation of the reaction conditions. The model for optimal reaction conditions of a lower antigenicity of a-casein and b-casein was established. Under the range of conditions studied, enzyme-to-substrate ratio had the most significant effects on the antigenicity of a-casein and b-casein. The anti-a-casein IgG binding inhibition and anti-b-casein binding inhibition were both significantly negatively related with the degree of hydrolysis (DH).

Keywords: cow’s milk allergy; a-casein; b-casein; hydrolysis; papain; ELISA; antigen; enzyme

1. Introduction

Food allergy has become a major public health concern around the world. Cow’s

milk is thought to be one of the most common food allergies (Fritsche, 2003). A

number of studies has presented that cow’s milk protein allergy (CMPA) has an

incidence of 2�6% for infants or young children (Hill & Hosking, 1996, 1997; Hosking, Heine, & Hill, 2000), causing serious consequences for the infants’ health.

Therefore, it is very necessary to reduce or eliminate the milk allergens. All milk proteins appear to be potential allergens, even those present in only trace

amounts. However, the main allergens in cow’s milk protein seem to be casein, a-LA and b-LG (Wal, 1998). Up to now, great attentions have been paid on how to reduce the allergenicity of whey protein, technological approaches involve heat processing

(Bu, Luo, Zheng, & Zheng, 2009; Kleber & Hinrichs, 2007), enzymatic hydrolysis

(Wróblewska, Jedrychowski, Hajós, & Szabó, 2008; Zheng, Shen, Bu, & Luo, 2008),

fermentation (Bu, Luo, Zhang, & Chen, 2010), Maillard reaction (Bu, Lu, Zheng, &

Luo, 2009; Bu, Luo, Lu, & Zhang, 2010) and high pressure (Kleber, Maier, &

Hinrichs, 2007). Combined application of high pressure and hydrolysis is also used

(Chicón, López-Fandiño, Alonso, & Belloque, 2008). Enzymatic hydrolysis with

selected proteases is by far the most efficient process. Several investigators have

evaluated the immunogenicity and allergenicity of enzymatically hydrolysed casein.

*Corresponding author. Email: [email protected]

Food and Agricultural Immunology

Vol. 23, No. 1, March 2012, 69�82

ISSN 0954-0105 print/ISSN 1465-3443 online

# 2012 Taylor & Francis

http://dx.doi.org/10.1080/09540105.2011.604770

http://www.tandfonline.com

http://dx.doi.org/10.1080/09540105.2011.604770

http://www.tandfonline.com

Wróblewska, Jedrychowski, Szabo, & Hajos (2005) carried out experiments that

commercial sodium caseinate isolate (SCI) was hydrolysed with Alcalase, pronase

and papain in a two-step process (Alcalase�papain, pronase�papain and pronase� Alcalase) to determine the changes in the immunoreactivity of a-casein, b-casein and k-casein, finding that the two-step process was an effective method in the reduction of immunoreactivity of casein, however, allergenic epitopes were still present in all

peptide fractions. Hussein, Gelencse’r, Polga’r, & Hajo’s (2000) found that enzymatic

peptide modification with methionine enrichment seems to be an efficient method for

the reduction of the potential allergenic character and for the improvement of the

nutritive value of buffalo and cow milk caseins. Wróblewska, Jedrychowski, & Farjan

(2007) used Alcalase, pepsin and lactozyme in a multi-step hydrolysis (Alcalase

followed by pepsin and next lactozyme) to determine the allergenicity of a low

molecular fraction of whey protein and sodium caseinate hydrolysates and found it

did not decrease the allergenicity of both whey protein and sodium caseinate

hydrolysates.

However, fewer studies were reported to predictive the effects of various

hydrolysis parameters on the antigenicity of casein hydrolysates. Therefore, the aim

of this study was to evaluate the effects of pH, temperature, enzyme-to-substrate

ratio and reaction time on the antigenicity of casein hydrolysates prepared by papain,

optimising the reaction conditions using the response surface methodology (RSM).

2. Materials and methods

2.1 Materials

The antigen proteins used for sensitisation studies and enzyme-linked immunosor-

bent assay (ELISA) were a-casein (C-6780; purity �90%) and b-casein (C-6905; purity �95%) purchased from Sigma Chemical Company (St. Louis, MO, USA).

Commercial casein obtained from Beijing Bio-technology Company (Beijing, China)

was used as hydrolysis reaction substrate. Papain used for the hydrolysis of casein

was purchased from FangShan Enzyme Preparation Factory (Beijing, China).

2.2 The preparation of rabbit antiserum

Rabbits for the sensitisation studies were 4�5 months old at the start of the study and weight was about 2.0 kg. Rabbits were sensitised with bovine a-casein (Sigma, C- 6780) or b-casein (Sigma, C-6905) as shown in Table 1. Seven days after the fourth sensitisation, blood samples were obtained from the rabbit hearts. Blood samples

were incubated for 1 h at room temperature and left overnight at 48C, then centrifuged for 10 min at 3000 �g to obtain sera. The sera were stored at �808C until the following analyses on the antigenicity of a-casein and b-casein by indirect competitive ELISA.

2.3 Proteolytic activity of protease

Procedures refer to folin phenol reagent method (Lowry, Rosebrough, Farr, &

Randall, 1951).

70 X. Liu et al.

The proteolytic activity of protease was determined by folin phenol reagent

method. The protease activity of papain used in the experiment is about 98253 units g �1

protein.

2.4 Casein hydrolysis

Coded and uncoded settings of the independent variables for casein hydrolysis according to central composite rotatable design are presented in Table 2.

Casein was hydrolysed with papain under the conditions which were shown in

Table 3. Prior to hydrolysis, casein was dissolved in 0.067 mol/L phosphate buffer,

stirring for 15 min at the pre-treatment temperature of 508C. During hydrolysis, pH was maintained by the addition of 1 M NaOH and 1 M HCl. The hydrolysis process

was terminated by heating the solution at 1008C for 10 min and then cooling immediately. The DH was determined according to the trinitrobenzene sulfonic acid

(TNBS) method (Nissen, 1979).

2.5 Estimation of antigenicity of casein hydrolysates by indirect competitive enzyme- linked immunosorbent assay (ELISA)

Specific procedures of indirect competitive ELISA refer to the method used earlier

(Zheng et al., 2008).

The residual antigenicity of casein hydrolysates was estimated by indirect

competitive ELISA. Microtitre plates with 96 wells (flat-bottomed; Costar, Corning Inc., Corning, NY, USA) were coated with a-casein (or b-casein) which

Table 1. Immunisation procedure of New Zealand rabbits.

Sequence Intervals(days) Antigen sensitisation

Dose (mg protein/

kg weight) Treatment

First 0 Antigen/Freud complete

adjuvant

0.5 Crural

intramuscular

Second 14 Antigen/Freud

incomplete adjuvant

0.5 Dorsal

subcutaneous

Third 10 Antigen 1.0 Otic plexus

Fourth 10 Antigen 1.0 Otic plexus

Table 2. Coded and uncoded settings of the independent variables for casein hydrolysis

according to central composite rotatable design.

Independent variables

Coded level pH Temperature(8C) E/S(%) Reaction time(min)

2 6.80 60.0 3.0 140

1 6.40 55.0 2.5 115

0 6.00 50.0 2.0 90

�1 5.60 45.0 1.5 65 �2 5.20 40.0 1.0 40

Food and Agricultural Immunology 71

was diluted in 50 mM carbonate buffer (pH 9.6) at 4 mg mL�1 (at 2 mg mL�1 for b-casein) and incubated overnight at 48C. In test tubes, 1 mg/mL solutions of various casein hydrolysates were incubated overnight at 48C with an equivalent volume of rabbit anti-a-casein or anti-b-casein antiserum diluted in 10 mM phosphate-buffered saline (PBS, pH 7.4) containing 1% BSA and 0.1% Tween 20

(PBS�BSA�Tween 20) (1:240,000 for anti-a-casein antiserum and 1:120,000 for anti-b-casein antiserum). The plates were washed four times the next day with 10 mM PBS containing 0.05% Tween 20 (PBS-T). After washing the plates, all wells

were filled with 100 mL per well of PBS�BSA�Tween 20 in order to block residual free binding sites, and incubated for 1 h at 378C. The plates were washed and then 100 mL per well of reactive mixtures of casein hydrolysates and polyclonal rabbit antibodies (IgG) were added and incubated for 1 h at 378C. Meanwhile, the addition of 100 mL of individual anti-a-casein or anti-b-casein serum was taken for the noncompetitive model. After washing the plates, the wells were filled with

100 mLper well of horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG (diluted 1:10,000) in PBS�BSA�Tween 20. After incubation for 1 h at 378C, the plates were washed again and then 100 mL per well of 3,3’, 5,5’-tetramethylen- benzidine (TMB, Amresco) substrate solutions were immediately added. The

plates were incubated at 378C for 10 min. Finally, 50 mL per well of 2 mol/L H2SO4 were added to stop the reaction. Absorptions were read spectrophotome-

trically at dual wavelengths of 450 nm and 630 nm by Multiskan MK3 ELISA

plate reader (Thermo Labsystems, Franklin, MA, USA).

The residual antigenicity of casein hydrolysates was calculated as follows:

Inhibition rate ¼ðB0 � BÞ=B0 � 100% (1)

where B is the absorbance measured in the presence of casein hydrolysate and B0 is the absorbance measured in the absence of casein hydrolysate. Low inhibition

rates indicate low residual antigenicity of casein hydrolysate for a-casein and b-casein.

2.6 Experimental design

In the experimental design, pH (X1), temperature (X2), enzyme-to-substrate ratio

(X3) and reaction time (X4) were chosen as independent variables. The two

dependent Y (Y1 and Y2) variables were to evaluate the residual antigenicity of

casein hydrolysates. Y1 reflects the residual antigenicity of a-casein of casein hydrolysates and Y2 reflects the residual antigenicity of b-casein of casein hydrolysates. Central composite rotatable design (CCRD) was used to optimise

independent variables, which contained five levels (Table 2) for each independent

variable, coded as �2, �1, 0, �1 and �2. Table 2 shows the correspondence among coded values and actual values of independent variables. Table 3 illustrates

the complete central composite design, which consisted of 2 4

experiments for a

full factorial design plus 2 �4 star experiments and 12 centre experiments, resulting in 36 experiments.

72 X. Liu et al.

Table 3. Full factorial central composite design matrix for anti-a-casein and anti-b-casein IgG binding inhibition.

Independent variables a

Dependent variables

Assay pH T(8C) E:S(%) t(min)

Y1: Anti-a-casein IgG binding inhibition

(%)

Y2: Anti-b-casein IgG binding inhibition

(%) DH(%)

1 5.6(�1) 45(�1) 1.5(�1) 65(�1) 15.69 30.48 9.34 2 5.6(�1) 45(�1) 1.5(�1) 115(1) 14.14 27.23 11.58 3 5.6(�1) 45(�1) 2.5(1) 65(�1) 10.60 21.38 12.07 4 5.6(�1) 45(�1) 2.5(1) 115(1) 9.41 19.27 16.31 5 5.6(�1) 55(1) 1.5(�1) 65(�1) 13.76 26.96 13.73 6 5.6(�1) 55(1) 1.5(�1) 115(1) 12.86 25.34 9.98 7 5.6(�1) 55(1) 2.5(1) 65(�1) 9.75 19.33 15.60 8 5.6(�1) 55(1) 2.5(1) 115(1) 11.89 17.77 13.84 9 6.4(1) 45(�1) 1.5(�1) 65(�1) 19.84 38.71 8.71 10 6.4(1) 45(�1) 1.5(�1) 115(1) 15.86 31.14 10.28 11 6.4(1) 45(�1) 2.5(1) 65(�1) 12.24 24.39 11.51 12 6.4(1) 45(�1) 2.5(1) 115(1) 11.33 20.67 11.57 13 6.4(1) 55(1) 1.5(�1) 65(�1) 18.44 37.07 9.01 14 6.4(1) 55(1) 1.5(�1) 115(1) 17.11 33.11 7.68 15 6.4(1) 55(1) 2.5(1) 65(�1) 14.05 27.94 8.37 16 6.4(1) 55(1) 2.5(1) 115(1) 15.15 29.74 8.71

17 5.2(�2) 50(0) 2(0) 90(0) 13.02 25.64 9.08 18 6.8(2) 50(0) 2(0) 90(0) 18.76 36.99 7.63

19 6(0) 40(�2) 2(0) 90(0) 15.90 32.12 8.92 20 6(0) 60(2) 2(0) 90(0) 14.02 27.75 11.20

21 6(0) 50(0) 1(�2) 90(0) 23.53 39.17 9.17 22 6(0) 50(0) 3(2) 90(0) 12.52 25.04 11.01

23 6(0) 50(0) 2(0) 40(�2) 16.96 34.55 9.59 24 6(0) 50(0) 2(0) 140(2) 14.02 27.78 14.57

25 6(0) 50(0) 2(0) 90(0) 9.64 23.57 11.41

F o

o d

a n

d A

g ric

u ltu

ra l

Im m

u n

o lo

g y

7 3

Table 3 (Continued )

Independent variables a

Dependent variables

Assay pH T(8C) E:S(%) t(min)

Y1: Anti-a-casein IgG binding inhibition

(%)

Y2: Anti-b-casein IgG binding inhibition

(%) DH(%)

26 6(0) 50(0) 2(0) 90(0) 10.67 27.26 9.66

27 6(0) 50(0) 2(0) 90(0) 9.54 24.82 9.93

28 6(0) 50(0) 2(0) 90(0) 11.53 23.48 10.01

29 6(0) 50(0) 2(0) 90(0) 8.68 23.52 9.96

30 6(0) 50(0) 2(0) 90(0) 9.30 22.54 10.44

31 6(0) 50(0) 2(0) 90(0) 10.63 23.41 10.61

32 6(0) 50(0) 2(0) 90(0) 9.64 23.40 11.72

33 6(0) 50(0) 2(0) 90(0) 9.50 22.12 9.74

34 6(0) 50(0) 2(0) 90(0) 9.20 23.05 10.23

35 6(0) 50(0) 2(0) 90(0) 10.59 22.56 9.63

36 6(0) 50(0) 2(0) 90(0) 9.67 23.41 9.82

a Values in parentheses are the coded levels of independent variables

7 4

X .

L iu

e t

a l.

2.7 Statistical analysis

SAS 8.2 (SAS Institute Inc., Cary, NC, USA) was applied to analyse the

experimental data. Response surface analysis took into account the main, the

quadratic and the interaction effects, according to the following equation:

Y ¼ b0 þ X4

i¼1 bi xi þ

i¼1 bii x

2 i þ

iBj¼2 bij xi xj (2)

where b0 is constants of the model. bi, bii and bij are regression coefficients, respectively, for linear terms, quadratic terms and interaction terms of the model. xi is the independent variable in the coded value. Student’s-t test was employed to assess

the significance of the b-coefficients for each dependent variable. The level of

significance was defined at PB0.05. The ANOVA is applied to evaluate the

significance and the fitness of the model, as well as the effects of individual terms

and their interactions on the responses. The three-dimensional response surface plots

were drawn using the fit quadratic polynomial equations by SAS, holding two of the

independent variables at a constant value and varying the other two variables within the experimental range.

3. Results and discussion

3.1 Assessment on models of antigenicity of casein hydrolysates for four independent variables

Table 3 illustrates the experimental design and results. The results for the regression

analysis are shown in Table 4. The significance and the fitness of the model were

assessed by ANOVA, in addition, the effects of individual terms and their

interactions on the responses were evaluated by ANOVA. Table 4 (‘full model’)

revealed that several terms were not significant (P�0.05). The nonsignificant terms

were eliminated to fit the full second-order model. For a-casein, the linear effects of temperature and reaction time are not significant. However, the quadratic effects of temperature and reaction time are significant. So the linear terms should be retained

in the model. In addition, for b-casein, the linear effect of temperature is not significant, but the interaction effects between temperature and pH is significant. So

the linear terms of temperature should be retained in the model. This procedure

resulted in the second-order model for a-casein with eight regression terms and that for b-casein with eight regression terms, respectively (Table 4,‘fitted model’). Table 4 showed that P-values of the ‘fitted model’ for a-casein and for b-casein were 0.0001, respectively. The adjusted R

2 of the ‘fitted model’ for a-casein is 0.861 and that for b-

casein is 0.816. The small P-values and high adjusted R 2

values demonstrate that the

model could give a good description of the relationship between responses and

independent variables.

3.2 Inhibition of anti-a-casein IgG binding to a-casein by casein hydrolysates

The effects of four independent variables on the anti-a-casein IgG binding inhibition could be observed by regression coefficients (Table 4) and response

surfaces (Figure 1). It is presented that both linear and quadratic effects of pH on

Food and Agricultural Immunology 75

the anti-a-casein IgG binding inhibition were highly significant (PB0.01). The linear effect of temperature was not significant (P�0.05), however, the quadratic

effects of temperature were highly significant (PB0.01). For enzyme-to-substrate

ratio, both linear and quadratic effects were highly significant (PB0.01). The

linear effect of reaction time was not significant (P�0.05), however, the quadratic

effects of reaction time were highly significant (PB0.01). The interactive effects

among independent variables did not appear to be significant (P �0.05). Enzyme-

to-substrate ratio was with the highest absolute regression coefficient, indicating

that enzyme-to-substrate ratio was the most significant among four independent

variables.

Figure 1a presents the effects of pH (X1) and temperature (X2), where the

enzyme-to-substrate ratio and reaction time are set on constant values of 2% (w/w)

and 90 min, respectively. From Figure 1a, the anti-a-casein IgG binding inhibition decreases with the increase of pH up to 5.5�6.0, then the anti-a-casein IgG binding inhibition increases again. It indicates that when the pH is 5.5�6.0, the minimal anti- a-casein IgG binding inhibition exists. Lieske & Konrad (1996) hydrolysed a-LA and b-LG by papain and found enzymic availability was best for a-LA when pH is 3.5

Table 4. Regression coefficients for the regression model for prediction of anti-a-casein and anti-b-casein IgG binding inhibition.

Full model Fitted model

Anti-a-casein IgG binding

inhibition (%)

Anti-b-casein IgG binding

inhibition (%)

Anti-a-casein IgG binding

inhibition (%)

Anti-b-casein IgG binding

inhibition (%)

coefficient P

Intercept 9.883 23.595 9.883 24.183

Linear

pH 1.558 0.0001 3.238 0.0001 1.558 0.0001 3.238 0.0001

Temperature 0.005 0.984 �0.198 0.716 0.006 0.985 �0.198 0.725 (E:S) �2.304 0.0001 �4.075 0.0001 �2.304 0.0001 �4.075 0.0001 Time �0.520 0.080 �1.480 0.012 �0.521 0.103 �1.480 0.013

Quadratic

pH 2

1.134 0.0001 1.226 0.015 1.134 0.0002 1.226 0.017

Temperature 2

0.901 0.001 0.881 0.072 0.901 0.002

(E:S) 2

1.668 0.0001 1.424 0.006 1.668 0.0001 1.424 0.006

Time 2

1.034 0.0004 1.189 0.018 1.034 0.0006 1.189 0.020

Interactions

pH �temperature 0.441 0.216 1.369 0.0496 1.369 0.055 pH �(E:S) �0.230 0.514 �0.314 0.637 pH �Time �0.226 0.520 �0.307 0.645 Temperature �(E:S) 0.664 0.069 0.884 0.193 Temperature �Time 0.540 0.134 0.707 0.294 (E:S) �Time 0.556 0.123 0.676 0.316

Other statistics

R 2

0.909 0.867 0.861 0.816

F 15.05 0.0001 9.789 0.0001 20.921 0.0001 14.979 0.0001

76 X. Liu et al.

and enzymic availability was best for b-LG when pH �7.5. This suggests that papain may have different effects on different substrates; and for different purposes,

favourite conditions are different even using the same enzyme. The anti-a-casein IgG binding inhibition decreases with the increase of temperature up to 48�548C, then the anti-a-casein IgG binding inhibition increases again, which suggests that when the temperature is 48�548C, the minimal anti-a-casein IgG binding inhibition exists. Temperature that exceeds 558C was not effective for the reduction of antigenicity of a-casein. However, Zheng (2009) found that temperature had no effect on the antigenicity of b-LG but had a negative effect on the anti-a-LA IgG binding inhibition when hydrolysing whey protein by papain. This may be that

papain has different effects on different substrates.

The effect of pH (X1) and enzyme-to-substrate ratio (X3) is shown in the

response surface plot (Figure 1b) where the temperature and reaction time are set

on constant values of 508C and 90 min, respectively. From Figure 1b, the inhibition decreases initially with the increase of enzyme-to-substrate ratio and

then the inhibition increases again. The minimal inhibition exists when enzyme-

to-substrate ratio is about 2.5%. It may be that with the enzyme-to-substrate ratio

increasing to 2.5%, some allergenic epitopes were destroyed, resulting in the

reduction of the allergenicity. But if the enzyme-to-substrate ratio was too high

exceeding 2.5%, the hydrolysis may be too active, some allergenic epitopes that

hidden inside may expose outside, causing the increase of the allergenicity again.

(a) (b) (c)

(d) (e) (f)

Figure 1. Response surfaces of the anti-a-casein IgG binding inhibition (Y1) using the b-coefficients from the fitted model (Table 4). (a) The effect of pH (X1) and temperature (X2) on

the inhibition at E/S � 2% (w/w), reaction time 90min; (b) the effect of pH (X1) and E:S (X3) on the inhibition at a temperature of 508C, reaction time 90min; (c) the effect of pH (X1) and reaction time (X4) on the inhibition at temperature of 508C, E/S � 2% (w/w); (d) the effect of temperature (X2) and E:S (X3) on the inhibition at a pH � 6, reaction time 90min; (e) the effect of temperature (X2) and reaction time (X4)on the inhibition at a pH � 6, E/S � 2% (w/w); (f) the effect of E:S (X3) and reaction time (X4) on the inhibition at pH � 6, temperature of 508C.

Food and Agricultural Immunology 77

It is illustrated in Figure 1c the effect of pH (X1) and reaction time (X4),

where the temperature and enzyme-to-substrate ratio are set on constant values of

508C and 2%, respectively. It shows that when reaction time is 80�100 min and pH is 5.5�6.0, the minimal inhibition exists. If the hydrolysis last for a longer time, some hidden allergenic epitopes may be outside, causing the allergenicity

increasing again. On the other hand, that prolonging the reaction time may cause

some bitter peptides occur, it is not good for taste. It is better to control the

reaction time around 80�100 min. Figure 1d, Figure 1e, Figure 1f show, respectively, the effect of temperature (X2)

and E:S (X3), temperature (X2) and reaction time (X4), E:S (X3) and reaction time

(X4) on the anti-a-casein IgG binding inhibition. It can be comprehensively concluded from the three figures that the minimal inhibition of anti-a-casein IgG binding to a-casein exists when temperature is 48�548C, enzyme-to-substrate ratio (E:S) is 2.5% and reaction time is 80�100 min.

The effects of the four independent variables (pH, temperature, enzyme-to-

substrate ratio and reaction time) are different on the anti-a-casein IgG binding inhibition. Finally, the optimum values of the four independent variables to reach

minimum levels of inhibition for a-casein were as follows: pH � 5.60, T �508C, E/ S � 2.5% (w/w), reaction time � 90 min. Under the above hydrolysis conditions, the inhibition for a-casein was 9.05% and that for b-casein was 19.25%.

3.3 Inhibition of anti-b-casein IgG binding to b-casein by casein hydrolysates

As we can see in Table 4 and Figure 2, the linear effects of pH on the anti-b-casein IgG binding inhibition are highly significant (PB0.01), the quadratic effects of pH

were significant (PB0.05). Neither the linear effect nor the quadratic effects of temperature is significant (P�0.05). For enzyme-to-substrate ratio, both linear and

quadratic effects are highly significant (PB0.01). And both linear and quadratic

effects of reaction time are significant (PB0.05). The interactive effects between pH

and temperature appear to be significant (PB0.05). The effect of enzyme-to-

substrate ratio is most significant among four independent variables for inhibition of

anti-b-casein IgG binding to b-casein. The effect of pH (X1) and temperature (X2) on anti-b-casein IgG binding

inhibition (Y2) is presented in the response surface plot (Figure 2a), where the enzyme-to-substrate ratio and reaction time are set on constant values of 2% (w/w)

and 90 min, respectively. From Figure 2a, the anti-b-casein IgG binding inhibition decreases with the increase of pH, indicating that pH has negative effects on anti-b- casein IgG binding inhibition. Zheng (2009) studied the antigenicity of WPC[AQ2]

hydrolysates prepared by papain, the results suggested when pH is around 5.5, the

antigenicity of a-LA is minimal. So it may be that papain could perform well in acid environment. Temperature had negative effects on the anti-b-casein IgG binding inhibition. With the temperature increases, the anti-b-casein IgG binding inhibition decreases.

Figure 2b shows the effect of pH (X1) and enzyme-to-substrate ratio (X3) on the

anti-b-casein IgG binding inhibition (Y2), where the temperature and reaction time are set on constant values of 508C and 90 min, respectively. When the

78 X. Liu et al.

enzyme-to-substrate ratio is increased from 1% to 2.4%, the inhibition decreases

rapidly and when the enzyme-to-substrate ratio exceeds 2.5%, the inhibition starts to

change slightly. Therefore, relatively high enzyme-to-substrate ratio is advantageous

to reduce the inhibition for b-casein, but ratio exceeds 2.5% has no significant impact on the inhibition.

The effect of pH (X1) and reaction time (X4) on anti-b-casein IgG binding inhibition (Y2) is observed in Figure 2c, where the temperature and enzyme-to-

substrate ratio are set on constant values of 508C and 2%, respectively. It shows that the inhibition is low when reaction time is 80�100 min along with low pH.

Figure 2d, Figure 2e, Figure 2f present, respectively, the effect of temperature

(X2) and E:S (X3), temperature (X2) and reaction time (X4), E:S (X3) and reaction

time (X4) on anti-b-casein IgG binding inhibition (Y2). They suggest that temperature has negative effect on the inhibition; and when enzyme-to-substrate

ratio reaches 2.5% and when reaction time is 80�100 min, the minimal inhibition exists.

The optimum values of the four independent variables to yield minimum levels of

inhibition for b-casein are as follows: pH � 5.20, T �558C, E/S � 2.5% (w/w), reaction time � 90min. Under the above hydrolysis conditions, the inhibition for a- casein was 11.81% and that for b-casein was 18.83%.

(a)

(d)

(b) (c)

(e) (f)

Figure 2. Response surfaces of the anti-b-casein IgG binding inhibition (Y2) using the b- coefficients from the fitted model (Table 4). (a) The effect of pH (X1) and temperature (X2) on

the inhibition at E/S � 2% (w/w), reaction time 90min; (b) the effect of pH (X1) and E:S (X3) on the inhibition at a temperature of 508C, reaction time 90min; (c) the effect of pH (X1) and reaction time (X4) on the inhibition at a temperature of 508C, E/S � 2% (w/w); (d) the effect of temperature (X2) and E:S (X3) on the inhibition at a pH � 6, reaction time 90min; (e) the effect of temperature (X2) and reaction time (X4) on the inhibition at a pH � 6, E/S � 2% (w/w); (f) the effect of E:S (X3) and reaction time (X4) on the inhibition at pH � 6, temperature of 508C.

Food and Agricultural Immunology 79

3.4 The relation between the inhibition and the degree of hydrolysis (DH)

Figure 3 and Table 5 present the relation between the inhibition and the degree of

hydrolysis (DH). It can be observed from Figure 3 that the anti-a-casein IgG binding inhibition and DH are negatively related. An increase in DH results in a

reduction in the inhibition for a-casein. The Ra (Table 5) between anti-a-casein IgG binding inhibition and DH shows highly significant negative relation. For b-casein, the anti-b-casein IgG binding inhibition is also highly significant negative with DH. This may be that with the DH increasing, more antigen epitopes are destroyed, so

that the antigenicity reduces. However, many findings indicate that enzyme

specificity, rather than the DH seems to determine the residual antigenicity of

milk protein. Ena, van Beresteijn, Robben, & Schmidt (1995) found that the

reduction in antigenicity varied with the enzyme used, this may be due to the fact

that even if the DH is very high, the short peptides might contain the antigen

epitopes. Jost, Monti, & Pahud (1987) found that DH had no relationship with the

antigenicity. Zheng et al. (2008) concluded that the anti-a-LA IgG binding inhibition was significantly negatively related with the DH, while the anti-b-LG binding inhibition was not related with the DH. So we can conclude that the

hydrolysis is different for different enzymes because of the high specificities of the

enzymes, also the hydrolysis is different even using the same enzyme because of

different substrates.

4. Conclusion

Enzymatic hydrolysis with papain can reduce the antigenicity of a-casein and b-casein effectively, however, the antigenicity of casein was not completely eliminated.

Under the range of conditions studied, enzyme-to-substrate ratio had the most

influential effect to reduce the allergenicity of casein; pH was found to influence

inhibition of a-casein and b-casein to a less extent. RSM was adequate to optimise several independent variables of the hydrolysis process simultaneously, resulting in a

casein hydrolysate with minimum antigenicity for a-casein and b-casein. The model

R2 = 0.191

R2 = 0.3659

7 9 11 13 15 17 DH(%)

in h ib

iti o n (%

)

anti- -casein inhibition

Figure 3. The relation curve between anti-a-casein (or anti-b-casein) IgG binding inhibition and the DH. Anti-a-casein inhibition: anti-a-casein IgG binding inhibition; anti-b-casein inhibition: anti-b-casein IgG binding inhibition; � linearity (anti-a-casein IgG binding inhibition);---linearity (anti-b-casein IgG binding inhibition).

80 X. Liu et al.

was adequate to represent the actual relationship between responses and independent

variables. The anti-a-casein IgG binding inhibition and the anti-b-casein IgG binding inhibition were both highly significant negative with DH.

Acknowledgements

This work was supported financially by the National Natural Science Foundation of China (award numbers 30471224 and 30871817) and National Science and Technology Ministry of China (award numbers 2006BAD27B04 and 2006BAD04A06).

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**significant at 0.01.

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