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In vitro digestion of bovine and caprine milk by human gastric and duodenal enzymes

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Abstract

In vitro digestion was performed by human proteolytic enzymes on bovine and caprine individual milks. Two types of caprine milk were investigated: with high and low contents of αS1-casein (CN). In addition the influence of heating of the milk on digestion was examined. The digestion was performed in two steps using human gastric and duodenal juice. Protein and peptide profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Caprine milk proteins were digested faster than bovine milk proteins. This was confirmed by the degradation profile obtained for both cows’ and goats’ milk, and was most evident for β-lactoglobulin. Comparing the digestion of milk protein from two groups of goats, high and low in αS1-CN content, respectively, did not show significant differences. Heat treatment of milk had a strong and significant effect on the level of digestion. Raw milk was degraded faster than the heat-treated milk, and the effect of heating was different for bovine and caprine milk.
International Dairy Journal 16 (2006) 961–968
In vitro digestion of bovine and caprine milk by human gastric
and duodenal enzymes
Hilde Almaas
a,
, Anne-Laure Cases
a
, Tove G. Devold
a
, Halvor Holm
b
, Thor Langsrud
a
,
Lars Aabakken
c
, Tormod Aadnoey
d
, Gerd E. Vegarud
a
a
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P. Box 1432-A
˚s, Norway
b
Department of Nutrition, University of Oslo, P. Box 1046 Blindern, 0316 Oslo, Norway
c
University of Oslo, Gastrointestinal Endoscopy, Rikshospitalet, 0027 Oslo, Norway
d
Department of Animal and Aquacultural Sciences, Norwegian University of Life Sciences, P. Box 1432-A
˚s, Norway
Received 4 May 2005; accepted 7 October 2005
Abstract
In vitro digestion was performed by human proteolytic enzymes on bovine and caprine individual milks. Two types of caprine milk
were investigated: with high and low contents of a
S1
-casein (CN). In addition the influence of heating of the milk on digestion was
examined. The digestion was performed in two steps using human gastric and duodenal juice. Protein and peptide profiles were studied
by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Caprine milk proteins were
digested faster than bovine milk proteins. This was confirmed by the degradation profile obtained for both cows’ and goats’ milk, and
was most evident for b-lactoglobulin. Comparing the digestion of milk protein from two groups of goats, high and low in a
S1
-CN
content, respectively, did not show significant differences. Heat treatment of milk had a strong and significant effect on the level of
digestion. Raw milk was degraded faster than the heat-treated milk, and the effect of heating was different for bovine and caprine milk.
r2006 Published by Elsevier Ltd.
Keywords: Digestion; Human proteolytic enzymes; Caprine milk; Bovine milk; Genetic polymorphism; Heat treatment
1. Introduction
Milk proteins provide a major dietary source for
humans, supplying amino acids for the synthesis of
proteins and other nitrogen-containing compounds
(Munro, 1969;Millward & Pacy, 1995;Young & Pellet,
1989). In addition, some of these proteins contain bioactive
peptides released by hydrolysis that may affect the human
health. These effects include mineral binding, growth
factors, blood pressure reduction (Tome
´& Debabbi,
1998) and protective properties against different micro-
organisms and viruses (Meisel & Schlimme, 1996;Pihlanto
& Korhonen, 2003). The nutritional efficiency of milk
proteins clearly depends on the content of essential amino
acids that is delivered during the digestion of the proteins,
and the absorption in the gut of amino acids and peptides
released (Bos, Gaudichon, & Tome
´, 2000).
There has recently been an increased attention on cows’
milk allergy, particularly among infants (Paupe, Paty, de
Blic, & Scheinmann, 2001;Sampson, 2004). As a result,
alternative sources for milk have been asked for. This has
lead to an increasing interest in and demand for caprine
and equine milk. Milk from the goat differs from that of
the cow in the composition of many components, which
may influence the digestibility of the milk. The composition
and structure of the fat, for instance, is quite different in
both types of milk. Goats’ milk contains smaller fat
globules and higher amounts of short-chain fatty acids.
The naturally emulsified fat of goats’ milk is, from a human
health standpoint, much easier to digest (Haenlein, 1992).
Also, the protein composition and structure of milk of
these animals differ, again with possible consequences for
the digestibility. Although the general distribution of
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0958-6946/$ - see front matter r2006 Published by Elsevier Ltd.
doi:10.1016/j.idairyj.2005.10.029
Corresponding author. Tel.: +47 64945866; fax: +47 64965901.
E-mail address: hilde.almaas@umb.no (H. Almaas).
proteins is quite similar in the milk of the two species,
variations in both the whey and casein (CN) fractions
occurs. Focus on genetic polymorphism of b-lactoglobulin
(b-LG) and a-lactalbumin (a-LA) has revealed important
differences between the bovine and caprine milk (Hill et al.,
1997;Moatsou, Hatzinaki, Samolada, & Anifantakis,
2005;Trujillo, Casals, & Guamis, 2000). A recent study
demonstrated major differences between hydrolysis of
bovine and ovine b-LG by pepsin, due to small variations
in the tertiary structure (El-Zahar et al., 2005). The ovine
b-LG was hydrolysed faster. No comparative investiga-
tions have been performed on caprine and bovine whey
proteins so far.
Studies of the genetic polymorphism of bovine and
caprine CNs have shown that the composition of a-, b-,
and k-CN is different in the milk from the two species
(Buchberger & Dovc, 2000;Ikonen, Ojala, & Syvaoja,
1997;Trujillo et al., 2000). The polymorphism of a
S1
-CN
has received considerable interest due to its strong influence
on the technological properties of the milk. Bovine milk
has nine different genetic variants, however, type B is
dominant in Europe (Bech & Kristiansen, 1990;Lien et al.,
1999;McLean, Graham, Ponzoni, & McKenzie, 1984). For
caprine milk, eighteen different alleles are identified. Four
different levels of a
S1
-CN content in the milk have been
found, referred to as ‘‘strong’’, ‘‘medium’’, ‘‘weak’’ and
‘‘null’’. The strong variant contains about 3.6 g L
1
a
S1
-
CN, while the ‘‘null’’ variant completely lacks this protein.
These differences are due to deletions, substitutions and
insertions in the DNA, defective mRNA processing and
loss of mRNA stability (Chianese, Ferranti, Garro,
Mauriello, & Addeo, 1997;Martin, Ollivier-Bousquet, &
Grosclaude, 1999;Martin, Szymanowska, Zwierzchowski,
& Leroux, 2002). In the Norwegian Dairy Goat breed, a
remarkable high frequency (70%) is lacking the a
S1
-CN in
the milk (Vegarud et al., 1999).
Besides depending on the type and amount of protein
present in the milk, its digestibility may also be affected by
heat denaturation. It has been reported that the heat
stability of caprine milk is lower than that of bovine
milk (Morgan, Jacquet, Micault, Bonnin, & Jaubert, 2000).
The degree of denaturation is dependent on several factors
as heating temperature, time, pH, ionic strength and
the concentration of soluble calcium and phosphate. CNs
have an open and flexible structure, giving a rather
high stability towards heat treatment. However, it is
reported that heating up to 90 1C may induce changes in
the size of the CN micelles (Devold, Brovold, Langsrud, &
Vegarud, 2000;Singh & Creamer, 1992). When milk is
heated complex reactions take place between whey proteins
and CNs. Three types of aggregates are formed; CN
and whey protein aggregates, CN micelles coated with
whey proteins, and internal aggregates between the
different whey proteins (Singh, 1995;Singh & Creamer,
1992).
The goal of the present study was to compare the in vitro
digestion of the proteins in samples of milk using human
gastric and duodenal enzymes, with major focus on three
aims: (i) comparison of the degradation of proteins in
bovine and caprine milk; (ii) evalution of difference in
digestibility of caprine milk with high content of a
S1
-CN
and without this protein, respectively; (iii) the influence of
heat treatment of milk on the digestibility of the proteins in
bovine and caprine milk.
2. Materials and methods
2.1. Collection and preparation of milk samples
Milk from 16 goats was collected individually from the
university farm in the southern part of Norway, in addition
to milk from six genetically typed goats in northern
Norway. All animals were of the breed Norwegian Dairy
Goat, undergoing the same type of traditional feeding. The
goats were divided in to two groups: eight goats lacking the
a
S1
-CN in the milk (G0), and eight goats expressing high
amounts of a
S1
-CN (GS). Grouping was done by genetic
typing, or by analysis of the a
S1
-CN content in the milk by
isoelectric focusing (IEF). Milk from eight Norwegian red
cattle cows from the university farm was also obtained
individually.
Analyses were carried out on skimmed milk. Fat
was removed by heating for 20 min at 37 1C, followed
by centrifugation at 3100 g(Bench top Beckman
GPR Refrigerated Centrifuge, swinging bucket rotor
GH 3.7, Beckman Coulter, CA, USA) for 20 min.
After storage at 20 1C for 20 min, fat was removed by a
spatula.
The effect of heating was studied on mixtures of 20 mL
milk from each individual animal. These mixtures were
prepared by pooling samples of milk from goats with low
content of a
S1
-CN, from goats with high amount of a
S1
-CN
and cows, respectively. Each of the mixtures was divided
into three different fractions, two undergoing different heat
treatments, pasteurisation (72 1C, 15 s) and high heating
(100 1C, 1 min), while the third fraction was kept untreated
as a reference. Each mixture contained milk from
eight individual animals, and all measurements were
repeated twice.
2.2. Protein content
Determination of total nitrogen (TN) and non-casein
nitrogen (NCN) in the milk was performed by Kjeldahl
analysis (IDF, 1993) according to standard protocol
(Kjeltec 1035 Analyser, Tecator, Ho
¨gana
¨s, Sweden). CN
nitrogen was calculated as the difference between TN and
NCN. The conversion factor of 6.38 was used to obtain the
content of crude protein (CP) and CN. The NCN fraction
was obtained in skimmed milk, after precipitation of the
CNs, according to a modified method of Aschaffenburg
and Drewry, (1959), and Pierre, Michel, and Le Graet
(1995).
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H. Almaas et al. / International Dairy Journal 16 (2006) 961–968962
2.3. Human gastric and duodenal enzymes
Human proteolytic enzymes were obtained in the
activated state by collecting human gastric (HGJ) and
duodenal juice (HDJ) according to Holm, Hanssen,
Krogdahl, and Florholmen (1988). All gastric and duode-
nal enzymes used in this study were obtained from one
person. In brief, a three-lumen tube enabled both
simultaneous instillation of saline in the duodenum, and
aspiration of gastric and duodenal juice from the volunteer.
Saline (100 mL h
1
) was instilled close to the papilla of
Vater to stimulate the production of proteolytic enzymes,
and duodenal juice was aspirated some 18 cm distally.
Aspirates were collected on ice and frozen in aliquots.
Before further use the individual samples of HGJ and HDJ
were mixed into two batches to avoid differences in enzyme
activity between the samples.
Proteolytic activity in the HGJ was assayed according to
Sanchez-Chiang, Cisternas, and Ponce (1987). The pepsin
activity was measured with bovine haemoglobin at pH 3.0
as a substrate. In HDJ the concerted action of proteases
and peptidases named ‘‘Total proteolytic activity’’ was
assayed at pH 8.0 with CN as substrate according to
Krogdahl and Holm (1979). The reactions were stopped
after 20 min of incubation by addition of 10% TCA. After
centrifugation, absorbance at 280 nm of the trichloroacetic
acid soluble hydrolysis product was used as measure of
proteolysis. One unit (U) of enzyme activity is defined as
the amount of enzyme that gives an absorbance of 1.0 at
280 nm in 20 min at 37 1C.
2.4. Digestibility assay—pH drop method
A modified digestibility assay, in vitro protein digestion
(AOAC Official Method 982.30; Rasco, 1994), was
performed in two steps, using HGJ and HDJ. The
procedure developed to mimic a ‘‘normal digestion’’ in
the human gastro-intestinal tract consisted of two incuba-
tion periods, imitating both the human stomach and the
duodenum. Each period lasted 30 min at 37 1C. Previous
results showed that no new peptides were produced with an
extended reaction time (unpublished results). First, 10 mL
of skimmed milk acidified to pH 2.5 with 2 MHCl were
incubated with 50 mL (0.4 U) HGJ. Then the pH was
adjusted to 7.5 with 1 MNaOH, 400 mL (13.0 U) HDJ was
added and the mixture was incubated again with contin-
uous stirring. The change in pH in the milk during the
degradation with HDJ was measured every minute, and the
corresponding pH curves were plotted. Aliquots (0.5 mL)
were also taken out for gel electrophoresis at different
times during the incubation. To stop the proteolytic
reactions, samples were put on ice, frozen and freeze dried.
The assay was performed with individual milk samples
from eight animals of each group: two groups of goats, and
one group of cows. Each sample was run in duplicate, and
results are presented as the average of all 16 measurements
within each group.
2.5. Gel electrophoresis
Sodium dodecyl sulphate polyacrylamide gel electro-
phoresis (SDS-PAGE) was carried out to evaluate the
protein profile after each step of hydrolysis (PhastSys-
tem
TM
, Pharmacia Laboratory Separation Division, Amer-
sham Biosciences, Uppsala, Sweden). The assay was
performed according to standard protocols (Laemmli,
1970), using 20% acrylamide gels (PhastSystem
TM
Homo-
geneous 20 gels, Amersham Biosciences). The molecular
mass markers used were the low molecular weight standard
kit (LMW Calibration kit, Amersham Biosciences). Stain-
ing was performed according to standard procedure
(Amersham Biosciences). Gels from SDS-PAGE were
scanned, and the amount of protein was quantified by
analysis using Image Master 1D quantification software
(Amersham Biosciences). The amount of protein was
divided with the total protein content in the milk (see
Section 2.2), in order to be able to compare results between
different milk samples.
Genetic variants of the a
S1
-CN from individual goats
were determined by IEF using ultra thin (0.3 mm) urea
containing polyacrylamide gels according to a modified
method of Erhardt (1989) (Devold et al., 2000;Vegarud
et al., 1989). A mixture of ampholytes was chosen in order
to give a maximum resolution of the caprine a
S1
-CN-
complex; Ampholine pH 3.5–5.0, Pharmalyte pH 4.2–4.9
and Pharmalyte pH 5.0–6.0 (Amersham Biosciences) were
used in the ratio 3:4:1. Coomassie Brilliant Blue R-250 was
used for staining. The different a
S1
-CN-variants of goats
were identified according to lyophilised CN samples from
goats known to be strong or lacking the a
S1
-CN in the milk
(kindly provided by Prof. F. Grosclaude, INRA, France).
2.6. Statistics
Student’s ttests (two-sample, assuming equal variances)
were run to compare the protein and CN contents in the
different types of goats’ milk (assuming one-tail alter-
native). Differences were considered significant when p
values were less than 0.05, here and in the following
analyses. For the digestion studies the drop in pH during
the first 5, 10 and 30 min of hydrolysis with HDJ were
studied. The drop in pH was modelled as dependent on the
groups of goats and cows (in proc GLM of SAS) using the
model:
Drop in pH ¼mean þmilk group þerror:(1)
Milk group was either from goats lacking the a
S1
-CN (G0),
from goats expressing the a
S1
-CN (GS) or from cows. The
term ‘‘error’’ is the effect of each of the eight individuals in
a group, in addition to random error. Significances of pair-
wise milk-type comparisons, and the contrast of goat
versus cow were estimated.
When comparing the effect of heat treatment on
mixtures of milk samples from the eight individual animals
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H. Almaas et al. / International Dairy Journal 16 (2006) 961–968 963
in each group, this model was used:
Drop in pH ¼mean þmilk group þheating
þðmilk type heatingÞþerror:ð2Þ
Milk group was in this model 2 the group of which the milk
samples were mixed. The heating methods used were
pasteurisation, high heat treatment/sterilisation and no
heat treatment (raw milk). The term ‘‘error’’ is from
duplicate samples of the same bulk milk. Significances of
pair-wise milk-type or pair-wise heating comparisons were
estimated.
3. Results
3.1. Comparison between hydrolysates from individual
bovine and caprine milk by gel electrophoresis
The degradation products from cows’ and goats’ milk,
after treatment with human gastric (pH 2.5) and duodenal
(pH 7.5) enzymes were studied by SDS-PAGE. Both steps
of hydrolysis were carried out during 30 min at 37 1C. The
protein profile after the two digestion steps, for individual
samples of both bovine and caprine milk is illustrated in
Fig. 1. The amount of proteins in each lane was quantified
by Image Master 1D quantification software. Fig. 1 shows
that the digestion with HGJ caused a major degradation of
the native proteins in milk from both cow and goat (lane
C1 and G1). The CNs were rapidly hydrolysed in milk
from both species. This was also observed for a-LA, serum
albumin, immunoglobulins and lactoferrin (LF). However,
the degradation of b-LG in cows’ and goats’ milk differed.
Most of the b-LG remained undigested (100%) during the
first degradation step with HGJ in bovine milk, while in
caprine milk approximately 35% remained intact. After
this first degradation step, digestion of cows’ milk also
seemed to produce more low molecular weight peptides
than digestion of goats’ milk.
During the second enzymatic step (Fig. 1, lane C2 and
G2), further degradation of both CNs and whey proteins
were observed. The most obvious difference between cows’
and goats’ milk was the digestibility of b-LG. Data from
Image Master showed that only about 23% of b-LG
remained undigested in the caprine milk, while the amount
of native b-LG in bovine milk was 83% after treatment
with HDJ.
IEF was used to identify the expression of a
S1
-CN in
milk from individual animals. The results of the protein
profile of milk from the individual Norwegian goats
grouped as G0, GW and GS are shown in Fig. 2A.
Seventy percent of Norwegian goats seem to lack the a
S1
-
CN, and most Norwegian animals belong to the group G0.
Analysis of the total CN content in the milk (ttest, see
Section 2.6) revealed significant differences (p¼0:04)
between the two groups of goats, G0 and GS. However,
no significant difference in total protein content was found
between the groups (p¼0:12).
The degradation of milk containing high amount of a
S1
-
CN (GS) is shown in Fig. 2B. The digestion profile of the
proteins showed a fast degradation of the a
S1
-CN with
HGJ, while b-CN, k-CN and b-LG seemed to be degraded
ARTICLE IN PRESS
Fig. 1. SDS-PAGE (20%) of skimmed milk from cow and goat (low
amount of a
S1
-casein), digested with human gastric and duodenal
enzymes. Arrows indicate the major bands; immunoglobulins (IG),
lactoferrin (LF), serum albumin (SA), casein, b-lactoglobulin (b-LG),
a-lactalbumin (a-LA) and peptides. The wells contain: (C) native cows’
milk, (C1) cows’ milk hydrolysed with HGJ (30 min, 37 1C), (C2) cows’ milk
hydrolysed with HGJ (30 min, 37 1C) and HDJ (30 min, 37 1C), (G) native
goats’ milk, (G1) goats’ milk hydrolysed with HGJ (30min, 371C), (G2)
goats’ milk hydrolysed with HGJ (30min, 371C) and HDJ (30min, 37 1C).
Standard molecular weight markers are shown on each side of the gel.
Fig. 2. IEF of caprine milk with various expression of the a
S1
-casein. Gel
A shows three different levels of a
S1
-casein; no expression (G0), weak
expression (GW) and strong expression (GS). Gel B demonstrates the
degradation of the strong type (GS) by digestion with human proteolytic
enzymes. GS: native form, GS1: digested with HGJ (30 min, 37 1C), and
GS2: digested with HGJ (30 min, 37 1C) and HDJ (30 min, 37 1C).
H. Almaas et al. / International Dairy Journal 16 (2006) 961–968964
more moderately. Further hydrolysis with HDJ showed
that most of the CNs and the whey proteins were digested
except b-LG.
3.2. Comparison of digestion between individual caprine and
bovine milk by the pH-drop method
The degradation of caprine and bovine milk by human
proteolytic enzymes was compared using the pH-drop
method. To investigate the influence of a
S1
-CN content on
the degradation of caprine milk, the milk the G0 and GS
groups of goats were studied. The results for bovine milk
and the two groups of goats are presented in Fig. 3.
A faster drop in pH was observed for milk from the two
groups of goats compared with that for milk from cows.
After 30 min the drop in pH of the bovine milk was
significant different (p¼0:049) from that of caprine milk.
These results showed that human gastric and duodenal
enzymes degraded the caprine milk faster than the bovine
milk. The milk from goat group G0 showed a faster pH
drop within the first 5 min compared with the milk from
group GS. After 10 and 30 min the slope of the curves were
identical. No significant statistical differences were ob-
served between the milk from the two goat groups, G0 and
GS (see model 1, Section 2.6).
3.3. Effect of heat treatment on the digestion of bovine and
caprine milk
Two types of heat treatment were performed, low (72 1C,
15 s) and high (100 1C, 1 min), on mixtures of milk samples
from group G0 and GS goats and from cows. The effect of
degradation by HGJ and HDJ on raw, pasteurised and
high heated milk was studied (Fig. 4). The data showed a
significant difference between cows’ and goats’ milk
(po0:0001). However, no difference between the two
groups of goats’ milk was observed (applying model 2,
Section 2.6).
Results from the pH drop experiments showed that raw
milk from cows and goats was digested by the enzymes
faster than the heated milk, as shown by a ‘‘larger’’ drop in
ARTICLE IN PRESS
7.40
7.50
7.60
7.70
7.80
7.90
8.00
8.10
0 102030
Time
(
min
)
pH
Fig. 3. Decrease in pH during enzymatic treatment with human duodenal
juice (HDJ) on goats’ and cows’ milk at 37 1C. Three groups of milks were
compared; G0 goats’ milk lacking the a
S1
-casein (K), GS goats’ milk with
a high expression of the a
S1
-casein (J), and cows’ milk (*). Each curve
shows the mean of milk from eight individual animals.
0
0.1
0.2
0.3
0.4
0.5
First 5 minutes First 10 minutes First 30 minutes
First 5 minutes First 10 minutes First 30 minutes
Change in pH during digestion with HDJ
raw milk
pasteurized
milk
high heated
milk
0.1
0.2
0.3
0.4
0.5
0
Change in pH during digestion with HDJ
raw milk
pasteurized
milk
high heated
milk
(A)
(
B
)
Fig. 4. Decrease in pH during treatment with human duodenal juice
(HDJ) at 37 1C after 5, 10 and 30 min for cows’ and goats’ milk. Raw,
pasteurised (15 s, 72 1C) and sterilised/high heated (100 1C, 1 min) milk
samples were tested: (A) bulk cows’ milk, mixed from eight individuals
and (B) bulk goats’ milk, mixed from eight individuals.
H. Almaas et al. / International Dairy Journal 16 (2006) 961–968 965
pH (Figs. 4A and B). Statistical analysis of the data
obtained after 30 min hydrolysis showed significant differ-
ences between raw and heated milk from both cows and
goats (po0:0001). A significant effect of interaction
between the two variables, group of animal and heat
treatment, was also found (p¼0:0036). This implies that
the effects of heat treatment on the subsequent protein
degradation are different for the various groups of animals.
The data obtained for bovine milk also indicated a higher
degradation of the pasteurised milk than the high heated
milk. The type of heat treatment did not seem to have
any important impact on the degradation of protein in
caprine milk.
4. Discussion
Milk proteins represent an important dietary source for
humans, provided that they are digested suitably. The
present work has, to mimic the in vivo situation, focused
on the in vitro digestion of bovine and caprine milk by
enzymes from the human stomach and duodenum.
Previous work (unpublished results) has shown that
commercial pepsin, trypsin and chymotrypsin from pig
give rise to different peptide profiles after hydrolysis of
milk. Using gastric and duodenal enzymes will give
better knowledge about the degradation of milk in humans,
and can reveal important issues with regard to the
proteins in nutrition. The results obtained may be
relevant for development of easy digestable products for
consumer groups with special needs, such as infants,
athletes and the elderly. However, it should be kept in
mind that this is only an in vitro model system, and that
clinical studies will be needed in order to make any clear
conclusions.
A comparison of the protein patterns from SDS-PAGE
(Fig. 1), illustrates the major protein profile after degrada-
tion of bovine and caprine milk by human proteolytic
enzymes. Most noticeable is the difference observed with
b-LG. After treatment with both HGJ and HDJ, image
analysis of the gel showed that only small amounts
(approximately 23%) of the caprine b-LG still remained
undigested, while the amount of intact bovine b-LG was
about 83%. Similar results from hydrolysis of ovine and
bovine b-LG by commercial pepsin were obtained by
El-Zahar et al. (2005). Ovine b-LG was degraded faster due
to its tertiary structure, and also surface hydrophobicity,
being slightly different from those of bovine b-LG
(El-Zahar et al., 2005). In order to confirm this for caprine
b-LG, further investigations will be needed. The studies on
the digestion of milk from both bovine and caprine
individuals with the pH drop method confirmed the
differences. Caprine milk was digested significantly faster
than bovine milk. The reason for this may probably be due
to the more resistant b-LG in bovine milk. However, other
variations in protein composition between bovine and
caprine milk could also contribute to this effect. Other
factors like variations in the tertiary structure due to amino
acid differences and genetic polymorphism may also
influence the result. An additional possibility is that the
enzymatic activity of the human juice may differ slightly in
the milk from the two species, due to variations in
the amount of minerals, carbohydrate content, buffer
capacity, etc.
IEF (Fig. 2) of individual milk samples showed different
levels of a
S1
-CN in the milk. It was demonstrated that a
S1
-
CN was partly degraded by HGJ, and then totally
hydrolysed with HDJ. This indicates that milk with a high
level of a
S1
-CN might take a longer time to be degraded
than milk lacking the protein. However, the results
demonstrated no significant difference in pH-drop between
the two groups of goats. The study showed an apparent
variation between the individual goats in each group.
This variation was probably due to other differences in
between the chosen individuals, such as genetic factors,
feeding, stage of lactation and other seasonal effects
(Devold et al., 2000).
Results on digestion of both cows’ and goats’ milk
showed that heated milk in general was more resistant to
hydrolysis. This is probably due to structural changes in
the proteins caused by denaturation and aggregation of the
whey proteins during heat treatment. New linkages
between the k-CN and the b-LG could be formed (Singh,
1995;Singh & Creamer, 1992), and the potential for these
protein aggregates to be attacked by human proteolytic
enzymes is different from that for native protein in
raw milk.
The significant interaction between the two variables,
group of animals (cows, G0 and GS) and heat treatment,
also showed that heating influenced the bovine milk in a
different way than the caprine milk. Degradation of high-
heated and pasteurised cows’ milk differed, while various
heat treatments on goats’ milk did not result in differences.
This may be due to the variations in the protein
composition of bovine and caprine milk as discussed
previously. Other differences between milk from the
two species might be of importance as well, such as
variations in the content of salts and carbohydrates.
Further work in this field will be needed to make a clear
conclusion.
5. Conclusions
The present study on in vitro digestion by human
proteolytic enzymes of caprine and bovine milk proteins
has provided new knowledge. Human proteolytic enzymes
degraded milk proteins from goat more rapidly than those
from cow. Most noticeable was the difference observed in
digestibility of b-LG. No significant effect was observed
between the digestion of goats’ milk with a high level of
a
S1
-CN, and milk from typical Norwegian goats lacking
this protein. Raw milk was digested significantly faster
with human proteolytic enzymes than pasteurised and
high-heated milk. This was the case for both bovine and
caprine milk.
ARTICLE IN PRESS
H. Almaas et al. / International Dairy Journal 16 (2006) 961–968966
Acknowledgements
This work is a part of a cooperation project between the
Department of Protein Chemistry and Technology at
Central Food Technological Research Institute (CFTRI)
in India, and the Department of Chemistry, Biotechnology
and Food Science at the Norwegian University of Life
Sciences. The project was financed by The Norwegian
Agency for Development Cooperation (NORAD), a
directorate under the Norwegian Ministry of Foreign
Affairs (MFA). We would like to thank Dr. V. Prakash
and Dr. P. Kaul for their cooperation. Furthermore, we
would like to thank Sara Wingren at the Norwegian
University of Life Sciences, and Andrea Criscione from
Universita
´degli Studi Mediterranea (Reggio Calabria,
Italy) for technical assistance.
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Chapter
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Casein (CN) micelle size was determined in 2 goat milks from animals with different genotype for αauthorCN. One milk lacked αauthor CN, the other had an αauthor CN content of 4.7 g kg-1. In comparison, the goat bulk milk has 3.3 g kg-1 αauthorCN. The mean diameter of micelles in milk without αauthor CN was found to be 280 nm, while the diameter was 199 nm in milk with a high content of αauthor CN, ie a decrease by 29%. Micellar sizes were estimated by photon correlation spectroscopy (PCS) and by transmission electron microscopy (TEM) associated with negative staining. Micellar volume distribution according to size showed that milk without αauthor CN had the typical distribution of goat milk, ie a large spread of diameters from 20 to 270 nm without any clear maximum in frequency and a high proportion of casein forming the large micelles. In contrast, milk with a high level of αauthor CN showed a narrow unimodal distribution of frequency. The chemical analysis of milks, total casein, individual caseins, total and soluble minerals showed that the only difference is the αauthor CN level. The results might imply a role of αauthor CN in micelle size regulation.