International Dairy Journal 9 (1999) 813}816

Resistance of b-lactoglobulin-bound lactose to the hydrolysis

by b-galactosidase
Franc7 ois Morgan, GweH naeK le Henry, Yvon Le GraeK t, Daniel MolleH ,
JoeK lle LeH onil, SamK d Bouhallab*
Institut National de la Recherche Agronomique, Laboratoire de Recherches de Technologie Laitie% re, 65 rue de Saint Brieuc, 35042 Rennes cedex, France
Received 21 July 1999; accepted 1 December 1999

Abstract

An in vitro study was conducted to investigate the sensitivity of lactose-b-lactoglobulin conjugates to b-galactosidase from
Kluyveromyces lactis. The hydrolysis was monitored by ion exchange chromatography. Compared to free lactose or lactulose, which
were rapidly hydrolysed, protein-bound lactose was not hydrolysed by b-galactosidase even after an extended hydrolysis time. Tryptic
digestion of the conjugates before addition of b-galactosidase improved the substrate accessibility and led to the release of about 50%
of the linked lactose. The results strongly suggest that the resistance of protein bound lactose is linked to the globular and compact
structure of lactose-b-lactoglobulin conjugates. ( 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Lactose-b-lactoglobulin conjugates; Hydrolysis; b-Galactosidase

1. Introduction highly reactive towards lactose (Burr, Moore & Hill,

Maillard reaction constitutes the major non-enzymatic
modi"cation of milk proteins subjected to heat process-
ing and storage (van Boekel, 1998). The early stage of the
reaction involves the condensation of the carbonyl group
of lactose (glucose moiety) with protein amino groups to
form a reversible Schi! base, which subsequently rear-
ranges to produce a more stable ketoamine. The extent of
the Maillard reaction in milk products depends on the
severity of heating and the length of storage period. The
consequences of the chemical glycation on the functional
as well as on the nutritional properties of proteins have
been extensively studied (O'Brien, 1995). Detrimental nu-
tritional e!ects include: (i) decrease or loss of the bio-
availability of essential amino acids such as lysine and
other nutrients, (ii) formation of toxic compounds, and
(iii) impairment of protein susceptibility and digestibility
by proteolytic enzymes (Sheer, Lee & Jelesciewicz, 1989).
Although this chemical reaction potentially involves
all the milk proteins, recent studies have reported that,
under mild heat treatment, b-lactoglobulin (b-LG) is
* Corresponding author.
E-mail address: said.bouhallab@rennes.inra.fr (S. Bouhallab)
1996; LeH onil, MolleH , Fauquant, Maubois, Pearce,
& Bouhallab, 1997). We have further described the struc-
tural changes induced by the glycation of b-LG. The
native-like conformation of the protein was preserved
when the glycation experiments were performed under
dry-way conditions (Morgan, LeH onil, MolleH & Bouhal-
lab, 1999).
Using dry-way glycated b-LG, the present study was
designed to determine the sensitivity of bound lactose to
the hydrolysis by b-galactosidase (b-Gase). The hydroly-
sis reactions were monitored by ion exchange
chromatography.
2. Materials and methods
2.1. Materials
b-LG B was prepared from the milk of homozygous
cows as previously described (LeH onil et al., 1997). The
freeze-dried b-LG B powder contained 93.2% proteins
and 0.6% lactose. b-LG represented 98% of the protein
content and a-lactalbumin less than 2%, based on rever-
sed-phase HPLC analysis. The b-LG concentration was
determined at 280 nm, using an E1{ of 9.6. Monohy-
1#.
drated lactose, glucose and galactose were purchased

0958-6946/00/$ - see front matter ( 2000 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 5 3 - 3
814 F. Morgan et al. / International Dairy Journal 9 (1999) 813}816
Nomenclature
b-LG b-Lactoglobulin
b-Gase b-Galactosidase
ONPG o-Nitrophenyl-b-D-galactopyranoside
from Merck (Darmstadt, Germany); lactulose, soybean
trypsin inhibitor and o-nitrophenyl-b-D-galactopyrano-
side (ONPG) from Sigma Chemical Co. (St. Louis, MO);
Tosyl-phenylalanine-chloromethylketone-treated tryp-
sin (E.C. 3.4.21.4; bovine pancreas; 31 U mg~1) from
Serva (Heidelberg, Germany). b-Gase (E.C. 3.2.1.23) from
Kluyveromyces lactis was supplied by Gist Brocades (Sec-
lin, France). Its activity at 373C, determined on ONPG in
0.1 M potassium phosphate bu!er pH 6.7 containing
1 mM MgCl and 0.87% b-mercaptoethanol, was
2
1460 U mL~1 of enzyme solution.
2.2. Preparation of samples
2.2.1. Glycation experiments
The dry-way glycation experiment was performed by
dissolving b-LG B (150 lM) in a 15 mM lactose solution
and adjusting the pH to 7.2 with 0.5 M NH OH. After
4
being freeze-dried, the protein-sugar powder was kept
under 65% relative humidity (saturated KI solution) and
503C for 12 h. The powder was then dissolved in cooled
distilled water to obtain a 150 lM b-LG solution and free
lactose was removed by extensive dialysis at 43C (50 mL
samples in 5 L distilled water containing 0.02% sodium
azide, 4 water batches in 72 h) in Spectra/Port 1 cellulose
membranes (molecular mass cut-o!: 6}8 kDa, Spectrum,
Laguna Hills, CA). The control sample was performed
using the same procedure without added lactose. The
dialysed samples were precipitated at pH 4.6 with 1 M
HCl at 233C for 1 h, "ltered on a cellulose acetate
0.45 lm Nalgenet "lter (Polylabo, Strasbourg, France)
and the pH of the "ltrate was adjusted to 7.2 with 2 M
NH OH. The "ltrates were freeze-dried and the powders
4
obtained were kept at 43C until further use. The average
number of lactose linked per monomer, measured by
electrospray ionization mass spectrometry as previously
described (Morgan, LeH onil, MolleH & Bouhallab, 1997)
was 3.81 (mol mol~1) in the glycated b-LG.
2.2.2. Tryptic hydrolysate of glycated b-LG
b-LG (150 lM) was dissolved in 50 mM potassium
phosphate bu!er pH 7.3 and hydrolysed with trypsin at
an enzyme/substrate ratio of 1/150 (w/w) at 373C for 3 h.
The reaction was stopped by cooling and by adding
Soybean Trypsin Inhibitor at an enzyme/inhibitor molar
ratio of 1/5.
2.3. Hydrolysis of free and bound lactose
with b-galactosidase
Hydrolysis experiments were performed with 4.4 or 22
units of b-Gase at 373C in 50 mM potassium phosphate
bu!er, pH 6.7. The following samples were studied: Free
lactose (292 lM); b-LG-lactose conjugate (76 lM protein,
292 lM lactose) and its control b-LG (76 lM)#free lac-
tose (292 lM); tryptic digest of glycated b-LG (76 lM
protein, 292 lM lactose) and tryptic digest of b-LG
(76 lM)#free lactose (292 lM).
Aliquots were withdrawn at various intervals and the
reaction was stopped by heating at 753C for 5 min,
cooled and diluted with MilliQ water before chromato-
graphic analysis.
2.4. Ion exchange chromatography
Lactose hydrolysis experiments were monitored by
anion-exchange chromatography on a Dionex system
(Dionex, Jouy en Josas, France) including Dionex DX500
apparatus, amperometric detector ED 40, and CarboPec
PA1 analytical column (4 i.d.]250 mm). Elution was
performed at 203C at a #ow rate of 1 mL min~1 by
mixing 0.2 M NaOH (solution B) in MilliQ water (solu-
tion A) using the following pH gradient: 8% solution
B from 0 to 15 min, and 8}100% from 15 to 26 min.
Quanti"cation was based on analysis of standard solu-
tions of pure lactulose, lactose, glucose or galactose with
known concentrations.
All experiments were performed in duplicate. The de-
termined coe$cients of variation were lower than 5%.
3. Results
Fig. 1 shows a typical pro"le of the separation of
lactose and derived monosaccharides by ion exchange
chromatography. The kinetics of the hydrolysis of lactose
by b-Gase are shown in Fig. 2. Total conversion of
lactose was obtained after 15 and 60 min with 22 and 4.4
Units of enzyme, respectively. Concomitantly to lactose
disappearance, the expected concentration of both gener-
ated products, i.e. glucose and galactose was recovered
indicating that the formation of oligosaccharides
throughout the transferase activity of the enzyme, does
not occur under these experimental conditions. Further-
more, since lactulosyl-amino groups represent the
stabilised forms of lactose-b-LG conjugates, the sensitiv-
ity of lactulose to b-Gase was tested. Complete digestion
of lactulose was reached after a 15 min hydrolysis (data
not shown).
The higher concentration of b-Gase was chosen to
examine the enzyme ability to remove the galactose
residue from the ketoamine compounds formed by
attachment of lactose to b-LG. Analysis of samples as a
 F. Morgan et al. / International Dairy Journal 9 (1999) 813}816
Fig. 1. Separation pro"le of galactose (1), glucose (2) and lactose (3) by
anion-exchange chromatography obtained after 10 min hydrolysis of
lactose (292 lM) by K. lactis b-galactosidase (4.4 units) in 50 mM potas-
sium phosphate bu!er, pH 6.7.
Fig. 2. Time course hydrolysis of free lactose (292 lM) with 22 (A) and
4.4 (B) units of K. lactis b-galactosidase at 373C in 50 mM potassium
phosphate bu!er, pH 6.7. lactose (n); galactose (e); glucose (h).
function of hydrolysis time revealed that lactose-b-LG
conjugates were resistant to b-Gase even in the case of a
6 h prolonged hydrolysis (Fig. 3A). The observed appear-
ance of glucose and galactose was attributed to
contaminant free lactose (ca. 40 lM) which was not
815
Fig. 3. Time course hydrolysis of lactose (292 lM) by K. lactis
b-galactosidase (22 units). (A): Lactose bound to b-lactoglobulin. (B):
Free lactose in the presence of unglycated b-lactoglobulin (76 lM)
lactose (n); galactose (e); glucose (h). Hydrolysis experiments were
performed at 373C in 50 mM potassium phosphate bu!er, pH 6.7.
completely removed by dialysis of the glycated b-LG.
The resistance of linked lactose did not seem to be due to
an inhibition of the enzyme activity by b-LG since the
time course hydrolysis of free lactose in solution was not
modi"ed by the presence of the protein as a co-solute
(Fig. 3B). The observed resistance of linked lactose to
b-Gase hydrolysis might arise from steric hindrance ef-
fects. This was supported by the activity of the enzyme on
a tryptic hydrolysate of the lactose-b-LG conjugates
(Fig. 4). The residual contaminant lactose present
due to incomplete dialysis, detected in the tryptic
hydrolysate (ca. 40 lM), accounted for the release of
corresponding amounts of glucose and galactose.
However, the measured galactose concentration reached
ca. 200 lM after 270 min of hydrolysis, suggesting that
about 50% of the lactose linked to the tryptic peptides
was hydrolysed.
4. Discussion
We have previously reported the biochemical charac-
terisation of lactose-b-LG conjugates formed by an
amino-carbonyl reaction in both solution and dry sys-
tems (Morgan et al., 1999). In the present work, we have
determined the sensitivity to K. lactis b-Gase hydrolysis
at pH 6.7 of the glycoconjugates obtained in the dry-way.
Compared to free lactose or lactulose, lactose bound to
816 F. Morgan et al. / International Dairy Journal 9 (1999) 813}816
Fig. 4. Time course hydrolysis of lactose (292 lM) bound to tryptic
peptides of glycated b-lactoglobulin by K. lactis b-galactosidase (22
units). lactose (n); galactose (e); glucose (h). Hydrolysis experiments
were performed at 373C in 50 mM potassium phosphate bu!er, pH 6.7.
b-LG was not hydrolysed by the enzyme, although the
most lysine residues involved in the lactose binding are
exposed at the protein surface (Morgan, Bouhallab,
MolleH , Henry, Maubois & LeH onil, 1998). A few data are
available on the hydrolysis by b-Gases of lactose bound
to proteins. However, Shida, Takamizawa, Nagaoka,
Kushiro and Osawa (1994a) have reported that a treat-
ment of two glycoproteins, detected in a proteose pep-
tone fraction of heated milk with b-Gase from jack bean,
abolished their binding ability to an E. coli heat-labile
enterotoxin. These two glycoproteins were supposed to
be glycated forms of a-lactalbumin and b-LG. In a sub-
sequent work, the same authors have shown that a model
lactose-a-lactalbumin amino-carbonyl product also lost
this binding ability after the same enzyme treatment
(Shida, Takamizawa, Nagaoka, Tsuji & Osawa, 1994b).
These e!ects were ascribed to the ability of b-Gase to
remove the terminal galactose residue from the glycocon-
jugates. The discrepancy between these results and those
presented in this study might arise from a better accessi-
bility of lactose to the active site of b-Gase when the
sugar is linked to a-lactalbumin compared to b-LG.
Furthermore, Shida et al. (1994b) have performed the
glycation experiments by heating a-lactalbumin with lac-
tose at 603C in an aqueous solution. As far as the glyca-
tion of b-LG is concerned, such treatment, in contrast to
that carried out in a restricted water environment used in
the present study, greatly a!ects the native state of the
protein as it was demonstrated in our previous work
(Morgan et al., 1999). When such glycated b-LG in solu-
tion was submitted to b-Gase digestion, a slow but e!ec-
tive release of galactose was obtained (results not shown).
Hence, the sensitivity of protein bound lactose to b-Gase
could be attributed to a better accessibility of the substra-
te, i.e. galactose, which is favoured by the protein de-
naturation after a glycation in solution. The relevance of
the structural e!ects of the protein on b-Gase activity is
also stressed out by using the tryptic hydrolysate of
glycated b-LG as substrate. In this case, about 50% of
linked lactose was digested.
In summary, lactose linked to b-LG is resistant to the
action of b-Gase mainly because of the globular and
compact structure of b-LG-lactose conjugates. Better
accessibility of bound sugar can be recovered either by
proteolysis or heat denaturation of the conjugates prior
to addition of b-Gase.
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