Effect of water activity, temperature and pH on solid state lactosylation of β-lactoglobulin

International Dairy Journal 23 (2012) 1e8

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Effect of water activity, temperature and pH on solid state lactosylation of b-lactoglobulin Marianne K. Thomsen, Karsten Olsen, Jeanette Otte, Kirsten Sjøstrøm, Birgit B. Werner, Leif H. Skibsted* Department of Food Science, Faculty of Life Sciences, University of Copenhagen, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark

a r t i c l e i n f o

a b s t r a c t

Article history: Received 30 May 2011 Received in revised form 13 October 2011 Accepted 13 October 2011

Glycation of whey proteins leads to changes of the nutritional and functional properties of the proteins. Lactosylation of b-lactoglobulin was monitored under conditions varying with respect to temperature, water activity (aw), and pH. The rate of the overall lactosylation and the average lactosylation degree increased with temperature (50e70
C) and aw of 0.51e0.64, but were only slightly affected by reaction mixture pH (pH 5e7) before drying. The reaction seemed to occur in two phases, the transition between which was related to a lowering of the glass transition temperature of the system. Detailed kinetic analysis of the initial step showed that the pseudo first-order rate constants for formation of monolactosylated b-lactoglobulin in general were higher than the rate constants for formation of di-lactosylated b-lactoglobulin. However, the temperature dependencies of the two reaction steps were similar, corresponding to an activation energy of about 100 kJ mol1. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction During processing and storage of whey products, the whey proteins react non-enzymatically with reducing sugars forming Schiff bases and Amadori compounds, i.e., early products of the Maillard reaction. Thus, in all whey protein preparations, a certain fraction of the proteins contain covalently attached lactose (Holt et al., 1999). On one hand, lactosylation alters the composition and properties of the whey proteins, and should therefore be minimized during whey protein preparation. On the other hand, attachment of lactose or other carbohydrates to dairy proteins has been shown to confer antioxidant properties to the protein (Chobert, Gaudin, Dalgalarrondo, & Haertlé, 2006; Lee, Tseng, & Wu, 2001; Yajima, Onodera, Takeda, Kato, & Shiomi, 2007), and to improve their functional properties (Chevalier, Chobert, Popineau, Nicolas, & Haertlé, 2001; Medranoa, Abiracheda, Panizzoloa, Moynaa, & Añónb, 2009; Nacka, Chobert, Burova, Léonil, & Haertlé, 1998; Oliver, Melton, & Stanley, 2006). In both instances, it is important to be able to control the extent of lactosylation to tailor and manipulate the properties of the proteins. b-Lactoglobulin (b-LG), the major whey protein in bovine milk, contains 15 lysine residues and an N-terminal amino group, which can be lactosylated at various rates. All potential reactive amino groups have been shown to react with lactose in a dry system during

* Corresponding author. Tel.: þ45 3533 3221; fax: þ45 3533 3344. E-mail address: [email protected] (L.H. Skibsted). 0958-6946/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2011.10.008

storage, however, with varying reactivity as a function of time (Fenaille, Morgan, Parisod, Tabet, & Guy, 2004; Morgan et al., 1999b). It has been shown that lactosylation of whey proteins occurs at temperatures as low as 25e45
C (Broersen, Voragen, Hamer, & de Jongh, 2004), and that the rate of lactosylation increases with increase in temperature in the range 50e65
C (Broersen et al., 2004; French, Harper, Kleinholz, Jones, & Green-Church, 2002; Guyomarc’h, Warin, Muir, & Leaver, 2000; Lund, Olsen, Sørensen, & Skibsted, 2005). French et al. (2002) observed that in this temperature range, lactosylation in a dry state (water activity, aw, ¼ 0.65) occurred much faster and to a larger degree than lactosylation in solution. Up to 11 lactose moieties were attached at the highest temperature, with a variation in glycoforms ranging from 4 to 11 carbohydrate moieties. Even for the dry-state lactosylation, the water activity is a major determinant for the reaction, the rate of reaction being maximal at intermediate water activities (0.4e0.8) due to the dual effect of water acting as a reaction product (mass-law retardation) and increasing the mobility of the reactants (van Boekel, 2001; Labuza & Baisier, 1992; Morgan, Nouzille, Baechler, Vuataz, & Raemy, 2005; Saltmarch, Vagnini-Ferrari, & Labuza, 1981). The reaction rate is also dependent on pH, since several steps in the Maillard reaction are acidebase catalysed, and it has been shown that for modification of b-LG with monosaccharides and fructooligosaccharides, the rate increased with pH from 5 to 8 (Broersen et al., 2004; Trofimova & de Jongh, 2004). The rate of reaction for the lactosylation of a-lactalbumin, the other major whey protein in bovine milk, in solution was also found to increase

2

M.K. Thomsen et al. / International Dairy Journal 23 (2012) 1e8

with increasing pH (Lund et al., 2005). The effect of pH on the reaction between lactose and b-LG in the dry state is largely unknown. Although pH is not clearly defined for dry systems, the pH of the protein solution prior to drying has been observed to influence the rate of reaction during subsequent heating (Broersen et al., 2004; Trofimova & de Jongh, 2004). Another factor, which may be important for the lactosylation of dry powder, is the physical state of the sample. The inclusion of protein in glassy matrices formed by amorphous carbohydrate has been a well-known way to preserve and stabilize proteins, e.g., for use in pharmaceuticals (Arakawa, Prestrelski, Kenney, & Carpenter, 2001; Constantino, Curley, Wu, & Hsu, 1998). The presence of protein in the matrix further stabilizes the glass and delays crystallization of the carbohydrate (Constantino et al., 1998; Thomas, Scher, & Desobry, 2004). Crystallization of carbohydrate potentially increases the molecular mobility of the system, which eventually accelerates physico-chemical alterations of the sample (Thomas et al., 2004). The lactosylation of b-LG in solution as function of time was linear (at 60
C) only up to a point where 1 lactose molecule was attached (Czerwenka, Maier, Pittner, & Lindner, 2006). The authors therefore speculated that the attachment of one lactose molecule to b-LG may make the protein less prone to react with the next lactose molecule. Despite the very small structural changes induced by the dry-state lactosylation (Medranoa et al., 2009), this might also be the case for dry-state lactosylation of b-LG, since the lactosylation reaction between b-LG and lactose in whey protein concentrate in the dry-state (aw 0.2e0.4) proceeded non-linearly with time at both 35 and 60
C, as measured by the percentage of blocked lysine residues (Morgan et al., 2005). Therefore, there is a need to study the kinetics of the initial steps in the lactosylation process in more detail. The objective of the present study was to obtain detailed information on the extent of lactosylation of b-LG in a dry system, under conditions prevailing during spray-drying. This was studied using liquid chromatographyemass spectrometry (LCeMS), a technique which provides accurate and detailed information about the extent and nature of modified protein adducts. A quantitative characterization of each lactosylated protein adduct formed during the reaction was performed to obtain a detailed kinetic model of the initial reactivity of the lysine residues in order to evaluate the effect of intermediate temperatures and water activities at pH-values normal to milk and towards pI of b-LG where the lysine residues are fully protonated and thus supposed to be less reactive towards

A

Preparation

lactose (Lund et al., 2005). A molar ratio of lactose and protein of 100:1 was used to obtain pseudo first-order reaction conditions, and the values of water activities and temperatures were selected to provide a sufficient level of lactosylation at each temperature, whilst at the same time avoiding denaturation of the protein. 2. Materials and methods 2.1. Materials

b-LG was isolated from bovine milk according to the procedure described by Kristiansen, Otte, Ipsen, and Qvist (1998), except that the milk used contained both major genetic variants of b-LG resulting in a mixture of variants A and B in the purified preparation. Lactose monohydrate (AnalaRÒ) was purchased from VWR (Poole, UK). Sodium bromide and potassium iodide were from VWR (Fontenay sous bois, France). Potassium hydrogenphthalate and potassium dihydrogenphosphate were from Merck (Darmstadt, Germany). 2.2. Experimental design As outlined in Fig. 1, the experiment consisted of two steps: (A) the preparation of the dry reaction mixtures, and (B) incubation at defined conditions and times followed by sampling and analysis. For the preparation of the reaction mixtures, lactose and b-LG were dissolved (100:1, molar ratio) in 50 mM phosphate buffer (pH 6.0 or 7.0) or 50 mM hydrogenphthalate buffer (pH 5.0) at room temperature. The solutions were frozen overnight at 40
C, lyophilized at 50
C in a BOC Edwards Modulyo freeze dryer (Buch & Holm, Herlev, Denmark), and stored as dry powders in closed containers at 40
C until use (Fig. 1A). The samples were divided into small beakers and distributed into several desiccators containing saturated solutions of NaBr (aw ¼ 0.51) and KI (aw ¼ 0.64) to control the water activity. The desiccators were then placed at 50, 60 and 70
C (Fig. 1B) in a thermostated (0.5
C) oven (Holten/Heraus, Denmark) for 120e720 min depending on temperature and aw. At defined incubation times, samples were taken out from the desiccators and stored at 40
C until analysis by liquid chromatographyemass spectrometry (LCeMS) and differential scanning calorimetry (DSC). A sample containing pure lyophilized b-LG was used as control. The methods employed are highly reproducible and since emphasis was on changes with pH, temperature and aw

Lyophilization

β-LG:lactose -1:100 (mol:mol) pH = 6

pH = 5

a w = 0.51

T = 50 60 70 C t = 720 360 180 min

B

a w = 0.64

50 480

60 70 C 255 120 min

a w = 0.51

50 60 70 C 720 360 180 min

Dissolvation

pH = 7

a w = 0.64

50 60 70 C 480 255 120 min

a w = 0.51

50 60 70 C 720 360 180 min

a w = 0.64

50 60 70 C 480 225 120 min

Analysis

Fig. 1. Outline of the experimental setup from preparation of reaction mixture (A) through kinetic experiment at various controlled conditions to analysis by LCeMS (B). The times indicated below denote the incubation times during which aliquot samples were taken for analysis.

M.K. Thomsen et al. / International Dairy Journal 23 (2012) 1e8

for the various kinetic parameters, each kinetic experiment was performed only once, but with a high number of samples in each experiment. 2.3. Liquid chromatographyemass spectrometry analysis The lyophilized protein/lactose mixtures sampled after various times under defined temperature and water activity conditions were dissolved in water (w1 mg protein mL1) and analyzed by LCeMS using the Agilent 1100 MSD Trap (Palo Alto, CA, USA) according to the procedure described by Ardö, Lilbæk, Kristiansen, Zakora, and Otte (2007). In brief, 5 mL were injected, and the proteins were separated on a Zorbax 300 SB-C18 column (2.1  12.5 mm) using a gradient of acetonitrile in TFA and water with detection at 210 and 280 nm. On-line MS was performed in the positive mode with a nebulizer pressure of 345 kPa, a flow of nitrogen of 9 mL min1 and a drying temperature of 300
C. Scans from 50 to 2200 m/z were taken at the normal scan resolution and the target mass set to 1521 m/z.

3

water activity conditions and the simultaneous formation of monolactosylated b-LG (b-LG1) and subsequently higher lactosylation states (e.g., b-LG2) was modelled using a simple consecutive reaction sequence as outlined in reaction scheme (2) with initial concentration and concentration at time t as given for t ¼ 0 and t ¼ t: kobs;1

kobs;2

b-LG/b-LG1/b-LG2 t ¼ 0 t ¼ t

C0b-LG Cb-LG

0 Cb-LG1

0 Cb-LG2

In this scheme, the first step represents the formation of a Schiff’s base complex, b-LGeL1, between b-LG and the first lactose, L, and the following rearrangement to the Amadori product, b-LG1, as outlined in scheme (3): k1

k

2 b-LG þ L % b-LGeL1 / b-LG1

k1

2.4. Data processing To quantify and compare the results obtained for each sample taken from the reaction mixture, an average mass spectrum was generated from a fixed time section of the total ion chromatogram (Dretention time ¼ 3.7 min) including the peak for b-LG and all lactosylated forms of b-LG. This average mass spectrum was deconvoluted with the following parameter settings: adduct ion Hþ; low mass 18,000; high mass 23,000; abundance cut off 5%; max. charge 20; min. peaks in set 5; envelope cut off 75% and molecular mass agreement 0.05%. Assuming a similar ionization efficiency of all b-LG forms, the relative amount of each lactosylated species was quantified from the intensities of the peaks of the deconvoluted mass spectrum. The average degree of lactosylation, D, of each sample was calculated using formula (1):

D ¼

Pn i ¼ 0 i  Iðprotein þ i  lactoseÞ P n i ¼ 0 Iðprotein þ i  lactoseÞ

(1)

where I is the sum of the intensities of b-LG A and B for each lactosylated form, and i is the number of lactose units attached to the protein in each lactosylated form (Lee et al., 2001).

(2)

(3)

where k1 is the second-order rate constant of the condensation of the protein with lactose and k1 and k2 are the first-order rate constants of the dissociation reaction and the Amadori rearrangement, respectively. If the decrease in b-LG is followed, the overall reaction of the first step would reflect the formation kinetics of the Schiff’s base complex with the observed rate constant, kobs,1, as seen in scheme (4): kobs;1

b-LG þ L/b-LG1

(4)

as [lactose] >> [b-LG], the reaction is considered pseudo-first order with the rate expression described by formula (5):

dCb-LG ¼ kobs;1 Cb-LG ; where kobs;1 ¼ k1 ½L dt

(5)

However, if the formation of b-LG1 is followed, the kinetics would reflect both the initial equilibrium by formation of the Schiff’s base and the following Amadori rearrangement. The rate expressions for the two steps are described by formulas (6) and (7):

dCb-LG1 ¼ k2 Cb-LGeL1 dt

(6)

dCb-LG1 ¼ k1 Cb-LG ½L  ðk1 þ k2 ÞCb-LGeL1 dt

(7)

2.5. Differential scanning calorimetry measurements The glass transition temperature was determined by conventional DSC using a DSC 820, (Mettler Toledo, Schwerzenbach, Switzerland), which is based on the heat flux principle and cooled with liquid nitrogen. Calibration of heat flow and temperature was performed with indium as the standard (mp ¼ 156.6
C, Mettler Toledo calibration kit, ME 119442). The linearity of the calibration was verified with zinc (mp ¼ 419.5
C, Mettler Toledo calibration kit, ME 119442), decane (Tm ¼ 29.66
C), and cyclohexane (Tm ¼ 6.47
C). Approximately 10 mg sample taken from the lactose/b-LG reaction mixture were loaded into 40 mL aluminum DSC crucibles, which were immediately hermetically sealed and transferred to the DSC. Samples were scanned over an appropriate temperature range with the heating scan rate 5
C min1 (linear upscan) and an empty crucible as reference. All measurements were performed in triplicate. 2.6. Kinetic model The disappearance of native b-LG during reaction in the lyophilized lactose/b-LG mixture under defined temperature and

The formation of the Schiff base is approximated to be at steady state:

dCb-LGeL1 k1 C ½L z00Cb-LGeL1 ¼ dt k1 þ k2 b-LG

(8)

Formula (8) is inserted in formula (6):

dCb-LG1 k1 C ½L ¼ k1 ½LCb-LG ¼ kobs;1 Cb-LG ¼ k2 $ dt k1 þ k2 b-LG

(9)

as [lactose] >> [b-LG] and k1 « k2 (the reverse reaction is much slower than the formation of the Amadori product). Thus, the disappearance of native protein and the formation of the Amadori product b-LG1 will occur with the same rate. Subsequent reaction of b-LG1 with an additional lactose molecule in the second step of scheme (2) is represented by kobs,2 and further lactosylation was found to occur but was neglected in the kinetic modelling. As [lactose] >> [b-LG], the second reaction was

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M.K. Thomsen et al. / International Dairy Journal 23 (2012) 1e8

also pseudo-first order with the rate expression described by formula (10):

dCb-LG1 ¼ kobs;1 Cb-LG  kobs;2 Cb-LG1 dt

(10)

Formulas (11) and (12) were obtained by solving the differential equations of (5) and (10).

Cb-LG ¼ C0b-LG $ekobs;1 t þ y0 " Cb-LG1 ¼

kobs;1 C0b-LG

(11)

#

kobs;2  kobs;1

" kobs;1 t

e



kobs;1 C0b-LG

#

kobs;2  kobs;1

ekobs;2 t þ y0 (12)

The rate constants kobs,1 and kobs,2 were determined by fitting the parameters of formulas (11) and (12) to the points obtained from the deconvoluted mass spectra by plotting the concentration of the native and mono-lactosylated b-LG as a function of time. The parameter y0 was set to zero assuming that all native protein eventually will be lactosylated, and furthermore that all monolactosylated protein will react further to yield the di-lactosylated protein. Any subsequent reactions were neglected in the kinetic model by limiting the reaction time included in the calculation. The A and B variants of b-LG were moreover considered to have the same reactivity. 2.7. Statistical analyses Data were analyzed by means of a variance test (PROC GLM) and the means were compared using Duncan’s test. All statistical

A

analyses were carried out using the statistical analysis software SAS 9.1 (SAS Institute, Cary, NC, USA), with p < 0.05 used as a threshold of statistical significance. 3. Results and discussion An example of chromatographic and mass spectrometric data is given in Fig. 2. As shown in Fig. 2A, the peaks for the A and B variants of native b-LG (peaks 1 and 2) disappeared during incubation at 70
C and a broad peak containing variously lactosylated forms of b-LG appeared at shorter retention times (peaks 4 and 5). By deconvolution of the peaks in the average mass spectrum, the relative abundance of each lactosylated species of both variants of b-LG was obtained. Up to 11 lactose units were attached at the highest temperature and aw (Fig. 2B). Minor amounts of b-LG variants with even higher lactosylation degree might be present but will not appear in the averaged mass spectra. The B variant of bLG appeared slightly more abundant, also in the lactosylated state. However, no significant differences in the rate of lactosylation could be detected when results were calculated separately for each genetic variant. Therefore, in the following, the results are all based on the sum of the two variants. 3.1. Average degree of lactosylation as affected by reaction conditions The average number of lactose units covalently attached to b-LG as a function of time, temperature, pH and water activity is shown in Fig. 3. An overall lactosylation rate, v, was determined from the slope obtained by linear regression of the linear part of each curve in Fig. 3, and the resulting rates are listed in Table 1.

2

t = 0 min

B

-LG B

1

-LG A +1 lactose

t = 15 min

t = 30 min 3

+2

Intensity (TIC)

Abs280 nm (mabs)

t = 45 min 4 t = 60 min

+3

+4

+5

t = 75 min +6

+7

t = 90 min 5

+8

+9 +10 +11

t = 105 min

30

t = 120 min

15 0 38

40

Retention time (min)

42

18000

19000

20000

21000

22000

Deconvoluted mass

Fig. 2. LCeMS results from analysis of samples prepared at pH 6, and allowed to react at 70
C and aw ¼ 0.64 for the time intervals indicated. Panel A, reverse phase high performance liquid chromatography chromatograms. Peak numbers indicate: 1, b-LG A; 2, b-LG B; 3, mono-lactosylated b-LG; 4 and 5, b-LG lactosylated to higher degrees. Panel B, deconvoluted mass spectra obtained from the chromatograms as described in the Data analysis section.

M.K. Thomsen et al. / International Dairy Journal 23 (2012) 1e8

10

A

8 6 4

Degree of lactosylation, D

2 0 10

B

8 6 4 2 0 10

C

8 6 4 2 0 0

100

200

300

400

500

600

700

800

Time (min) Fig. 3. The average degree of lactosylation of b-LG at pH 5 (A), pH 6 (B), and pH 7 (C) as a function of reaction time. Reaction mixtures were prepared and allowed to react at the various conditions shown in Fig. 1: -, aw ¼ 0.64, 50
C; ,, aw ¼ 0.51, 50
C; C, aw ¼ 0.64, 60
C; B, aw ¼ 0.51, 60
C; :, aw ¼ 0.64, 70
C; 6, aw ¼ 0.51, 70
C.

Even heat treatment at 50
C (8 h) leads to a lactosylation degree of 4 at pH 7.0 and aw ¼ 0.64, which is in accordance with the results of Morgan, Léonil, Mollé, and Bouhallab (1999a), who found approx. 2.5 lactose molecules linked to each b-LG monomer. The lactosylation degree and reaction rate for lactosylation was significantly (p < 0.0001) increased with temperature, especially from 60
C to 70
C (Fig. 3). The highest lactosylation degree, an average of 8.5 lactose molecules attached to each b-LG molecule, as well as the highest rate of lactosylation, was obtained for the reaction

5

mixture prepared with pH 7 and allowed to react at 70
C and aw ¼ 0.64 for 120 min (Table 1). From the rates obtained at the three temperatures the energy of activation (Ea) for the overall reaction was obtained at varying pH and water activity. The values for the activation energy of around 100 kJ mol1 is in line with the results of Naranjo, Malec, and Vigo (1998), obtained by incubating lysine with various mono- and disaccharides. However, due to the large standard deviations, there were no significant differences between the Ea for the various conditions. Increasing pH during the preparation of the dry reaction mixtures resulted in an increase in the overall degree of lactosylation, as well as in the rate of lactosylation (p < 0.01), most markedly at the highest storage temperatures and water activities (Fig. 3AeC and Table 1). Reaction mixtures prepared at pH 5 became significantly less lactosylated compared with reaction mixtures prepared at pH 6 and 7 (p < 0.0005), which were almost identical with respect to the overall degree of lactosylation. During the drying process an amorphous matrix is formed in which the protein is dissolved in the carbohydrate, which replaces water as a dry “solvent” (Arakawa et al., 2001; Constantino et al., 1998; Le Meste, Champion, Roudaut, & Simatos, 2002). Since pH 5 is very close to the pI of b-LG (w5.4; Sawyer, 2003), it could be speculated that proteineprotein interactions would be substantial at this pH and thus it would be more difficult for free amino groups to react with lactose. Furthermore, the reactivity of these amino groups is limited at the low pH, where the amount of the reactive unprotonated amine is very low (Labuza & Baisier, 1992). When the pH increases, the protein becomes more negatively charged at the surface, causing charge repulsion and thus a decrease in proteineprotein interactions. In addition, the amount of reactive free amino groups increases and thus also the degree of lactosylation increases. Broersen et al. (2004) observed an almost linear increase in the rate of reaction between glucose and b-LG with pH in a dry matrix, when pH was increased from 5 to 8. In the reaction between oligofructose and b-LG an increase in pH from 7 to 8 resulted in a marked increase in the number of sugar molecules attached, especially at shorter incubation times (Trofimova & de Jongh, 2004). Although in the present study, the difference between the reaction rates at pH 6 and 7 was less pronounced (Table 1), it could be worthwhile considering the pH of the whey protein concentrate entering the spray tower with respect to control of the lactosylation degree as well as the functionality of the final product. 65 60 55

T storage

50 45

pH, aw

v (average lactose units attached min1)a





Ea (kJ mol1)b

40

T g ( C)

Table 1 Overall reaction rates and activation energies for lactosylation of b-LG at varying conditions.

35 30 25 20




50 C

60 C

70 C

pH 5, aw ¼ 0.51 pH 5, aw ¼ 0.64

0.003 0.005

0.007 0.011

0.034 0.050

106  13 99  14

pH 6, aw ¼ 0.51 pH 6, aw ¼ 0.64

0.004 0.009

0.006 0.016

0.047 0.071

100  39 84  21

pH 7, aw ¼ 0.51 pH 7, aw ¼ 0.64

0.005 0.010

0.008 0.017

0.049 0.073

98  32 83  21

15 10 5 0 0

a

v is obtained from linear regression of data shown in Fig. 3. Ea is obtained as R$slope from the plot lnv versus T1. Data represent slope  standard deviation from linear regression. b

100

200

300

400

500

600

700

Time (min) Fig. 4. The glass transition temperature, Tg, during reaction at 50
C and aw ¼ 0.51, of reaction mixtures prepared at different pH (-, pH 5; C, pH 6; :, pH 7). The storage temperature, T, is marked with the dotted line.

6

M.K. Thomsen et al. / International Dairy Journal 23 (2012) 1e8

ratio, types of carbohydrate and protein). Furthermore, it might be almost impossible to separate the effects of temperature and water activity on the rate of reaction, which is illustrated by a comparison of the reaction rates for reaction conditions of 60
C and aw ¼ 0.51 and of 50
C and aw ¼ 0.64, for which almost the same reaction rates and degrees of lactosylation were observed. The effect of water activity and temperature apparently counterbalance each other in the sense that the mobility in the matrix becomes comparable for these conditions.

A

B

3.2. Effect of the physical state of the sample matrix

Intensity

From a more careful inspection of Fig. 3 it becomes evident that the lactosylation reaction occurs in two phases. The progress of the reaction was very limited initially, in some cases even nonexisting, until a certain point after which lactosylation proceeded at an almost linear reaction rate in the second phase. The bi-phasic nature of the reaction was most pronounced at 50
C and at the lower water activity (aw ¼ 0.51), but could also be observed at other reaction conditions (Fig. 3). The observed tendency of the lactosylation reaction to proceed very slowly early in reaction could indicate that some sort of physical barrier had to be overcome, e.g., a glass transition, before the conditions for reaction between lactose and b-LG could proceed at a measurable rate. To test this hypothesis an investigation of the physical state of the selected dry reaction mixtures sampled at 50
C and aw ¼ 0.51 was performed using DSC. The results of these measurements are shown in Fig. 4, where the storage temperature is marked with a dotted line in order to illustrate the difference between storage temperature, T, and the glass transition temperature, Tg, during the course of the lactosylation reaction. The results in Fig. 4 indicate that a Tg was detected during the course of the lactosylation reaction for aw ¼ 0.51 and 50
C regardless of the initial pH. It was not possible to quantify the amount of lactose in the glassy state compared to crystallized lactose on the basis of the DSC measurements. However, it is clear that at least part of the dry reaction mixtures remained in a glassy state during the reaction at the actual temperature, which is somewhat higher than the Tg (w26
C at t ¼ 0 min). Interestingly, Tg decreased markedly after 180 min corresponding to the end of the observed lag phases for the reaction (see Fig. 3). The decrease in Tg indicates plastization, which is most likely due to water absorption by the glassy system further increasing mobility of the reactants, in effect creating the more optimal conditions for reaction between lactose and b-LG. Through the initial steps of the lactosylation reaction, water is released as a reaction product (van Boekel, 2001; Labuza & Baisier, 1992), which together with the water continuously absorbed from the surroundings, increases the mobility in the system, leading to a further increase in reaction rate, as is evident from Fig. 3.

C

0

100

200

300

400

500

Time (min) Fig. 5. Experimental values and model fits for the disappearance of the native b-LG (-), and the formation of singly lactosylated b-LG (C) as a function of time, in reaction mixtures prepared at pH 5 (A), pH 6 (B) and pH 7 (C) and stored at 50
C and aw ¼ 0.64. The curves were obtained by use of formulas (8) and (9). The formation of dilactosylated b-LG (:) is also shown.

Increasing the water activity from 0.51 to 0.64 resulted in significant increases in the rate of lactosylation at all pH values and reaction temperatures (p < 0.0001) (Fig. 3). This could in part be due to a lower Ea at the higher water activity (Table 1). Saltmarch et al. (1981) found the maximal rate of browning in whey powders at aw w 0.5, and in general browning is considered to be maximal in the water activity interval of 0.5e0.8 (Labuza & Baisier, 1992). However, it should be recognized that the different steps of the Maillard reaction may depend differently on the water activity (Bell, Touma, White, & Chen,1998), and that the reaction rate also depends on the constitution of the dry matrix (e.g., carbohydrate:protein

Table 2 Rate constants, kobs,1, for the disappearance of native b-LG and the formation of mono-lactosylated b-LG at various conditions. pH, aw

kobs,1 (min1)a


Ea (kJ mol1)b





50 C

60 C

70 C

pH 5, aw ¼ 0.51 pH 5, aw ¼ 0.64

0.0025  0.0002 0.0049  0.0006

0.0041  0.0004 0.0074  0.001

0.019  0.002 0.036  0.005

nd nd

pH 6, aw ¼ 0.51 pH 6, aw ¼ 0.64

0.0032  0.0003 0.0092  0.0004

0.0090  0.0005 0.0087  0.001

0.022  0.003 0.035  0.006

nd nd

pH 7, aw ¼ 0.51 pH 7, aw ¼ 0.64

0.0032  0.0005 0.0068  0.0006

0.0067  0.0006 0.0113  0.0008

0.021  0.003 0.046  0.006

86  10 90  22

a b

Data represent values  error from fit according to Equation (11). Ea is obtained as R$slope from the plot lnk versus T1. Data represent slope  standard deviation from linear regression; nd, not determined.

M.K. Thomsen et al. / International Dairy Journal 23 (2012) 1e8

7

Table 3 Rate constants, kobs,2, for the disappearance of mono-lactosylated b-LG at various conditions. pH, aw

kobs,2 (min1)a 50
C

60
C

70
C

pH 5, aw ¼ 0.51 pH 5, aw ¼ 0.64

0.0024  0.001 0.0045  0.001

0.0015  0.001 0.0067  0.002

0.019  0.010 0.029  0.013

nd nd

pH 6, aw ¼ 0.51 pH 6, aw ¼ 0.64

0.0015  0.001 0.0090  0.002

0.0045  0.002 0.0087  0.005

0.022  0.009 0.034  0.020

nd nd

pH 7, aw ¼ 0.51 pH 7, aw ¼ 0.64

0.0039  0.001 0.0049  0.003

0.0067  0.002 0.011  0.003

0.021  0.009 0.045  0.020

80  17 101  14

a b

Ea (kJ mol1)b

Data represent values  error from fit according to Equation (12). Ea is obtained as R$slope from the plot lnk versus T1. Data represent slope  standard deviation from linear regression; nd, not determined.

3.3. Detailed kinetics of the first lactosylation steps During the reaction in the lyophilized b-LG/lactose mixture, the disappearance of native b-LG and formation of mono-lactosylated b-LG followed a typical pattern of a consecutive reaction sequence over time, as depicted in Fig. 5. This is a typical behaviour for an intermediate like the Amadori product: at first a build-up of the component, followed by a slow or fast decrease (depending on temperature and the system), and thus, the kinetics of the involved species complied with formula (9). As shown in Fig. 5, the reaction of the mono-lactosylated b-LG results in generation of the second Amadori compound with two lactose molecules attached to the protein, in accordance with reaction scheme (2), followed by formation of higher lactosylation states at longer time scales (as seen in Fig. 2B). The resulting rate constants, kobs,1 and kobs,2 are listed in Table 2 and Table 3. The kinetic analysis did not include the intensity data for the di-lactosylated b-LG because of the induction period for this compound, which resulted in too few data points for an exponential fit. As for the average degree of lactosylation, it is expected that the reactivity of the amino group should increase with pH, resulting in increased reaction rates for disappearance of native b-LG from pH 5 to 7, because of a higher effective concentration of the unprotonated form of the 3-amino group of lysine. This tendency is most obvious considering the rate constants, kobs,1, at 50
C and 60
C at aw ¼ 0.64. However, at aw ¼ 0.51, it seems that some physical boundaries (barriers) are changing the individual chemical pathways making up the initial Maillard reaction since the kobs,1-values are not varying according to the normal activation profile of the mechanism. This also relates to kobs,2, but only at 50
C, which might be due to formation of the water in the first step of the reaction, leading to a decrease of the physical inertia controlling the kobs,1-values. However, statistical analysis of all the kinetic values showed no significant difference between kobs,1 and kobs,2 at various pH (p > 0.05). This result supports the explanation that the unprotonated amino form is only partly involved in the initial steps of the dry reaction mixtures. Both aw and temperature significantly affected the kinetics of both reactions controlled by the rate constants kobs,1 and kobs,2. The reactivity between the sugar and the amino group was highest at the highest values, i.e., 70
C and aw ¼ 0.64, respectively. Surprisingly, neither of the mean rate constants (kobs,1 and kobs,2) were significantly different at 50
C and 60
C, probably because of the decreased rate constant from 50
C to 60
C for the samples incubated at pH 6 and aw ¼ 0.64 (both rate constants), and at pH 5 and aw ¼ 0.51 (kobs,2). Due to a large standard deviation on the determination of the rate constants of the initial reactions at pH 5 and pH 6, Ea-values have been given for the reaction mixtures at pH 7 only. As seen in Tables 2 and 3 the kinetics of the initial steps of the

Maillard reaction are not controlled differently than the overall reactivity of the sugar and protein mixture, both resulting in an Ea of approximately 100 kJ mol1. 4. Conclusions For the reaction of b-LG with excess of lactose in the dry state under the present conditions, the lactosylation proceeded linearly with time, and the reaction rate for mono- and di-lactosylated b-LG was similar. However, a lag phase appeared at low temperature and aw due to a glass transition where the mobility of reactants were highly limited before water released during the initial steps of lactosylation increased the mobility of the reactants in the system. The rate of lactosylation was significantly affected by temperature, aw and pH, and the results of this study suggest that lactosylation of b-lactoglobulin may be controlled by decreasing the temperature used for drying, and to a lesser extent by reducing the pH before drying and by quickly lowering the water activity to below 0.5. Acknowledgement This work was supported by the Danish Dairy Research Foundation and the Danish Council for Independent Research j Technology and Production Sciences (FTP). References Arakawa, T., Prestrelski, S. J., Kenney, W. C., & Carpenter, J. F. (2001). Factors affecting short-term and long-term stabilities of proteins. Advanced Drug Delivery Reviews, 46, 307e326. Ardö, Y., Lilbæk, H., Kristiansen, K. R., Zakora, M., & Otte, J. (2007). Identification of large phosphopeptides from b-casein that characteristically accumulate during ripening of the semi-hard cheese Herregård. International Dairy Journal, 17, 513e524. Bell, L. N., Touma, D. E., White, K. I., & Chen, Y.-H. (1998). Glycine loss and Maillard browning as related to the glass transition in a model food system. Journal of Food Science, 63, 625e628. van Boekel, M. A. J. S. (2001). Kinetic aspects of the Maillard reaction: a critical review. Nahrung/Food, 45, 150e159. Broersen, K., Voragen, A. G. J., Hamer, R. J., & de Jongh, H. H. J. (2004). Glycoforms of b-lactoglobulin with improved thermostability and preserved structural packing. Biotechnology and Bioengineering, 86, 78e87. Chevalier, F., Chobert, J.-M., Popineau, Y., Nicolas, M. G., & Haertlé, T. (2001). Improvement of functional properties of b-lactoglobulin glycated through the Maillard reaction is related to the nature of the sugar. International Dairy Journal, 11, 145e152. Chobert, J.-M., Gaudin, J.-C., Dalgalarrondo, M., & Haertlé, T. (2006). Impact on Maillard type glycation on properties of beta-lactoglobulin. Biotechnology Advances, 24, 629e632. Constantino, H. R., Curley, J. G., Wu, S., & Hsu, C. C. (1998). Water sorption behavior of lyophilized protein-sugar systems and implications for solid-state interactions. International Journal of Pharmaceutics, 166, 211e221. Czerwenka, C., Maier, I., Pittner, F., & Lindner, W. (2006). Investigation of lactosylation of whey proteins by liquid chromatographyemass spectrometry. Journal of Agricultural and Food Chemistry, 54, 8874e8882.

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