Influence of high altitude and of forage from alpine origin on protein
composition and renneting properties of cow’s milk
F. Leiber, D. Nigg, C. Kunz, M.R.L. Scheeder, M. Kreuzer and H.-R. Wettstein
Institute of Animal Science, Animal Nutrition, ETH Zurich, CH-8092 Zurich, Switzerland
Summary
A two factorial design was applied to separate the effects of high altitude and alpine forage quality
on milk protein and cheese-making properties. Milk was analysed for protein composition, plasmin
and plasminogen content, and rennet coagulation properties. High altitude caused slightly decreased
milk protein contents, a decreased plasminogen content and significantly impaired rennet clotting
time. Hay of alpine origin, when compared to hay of lowland origin, led to significant lower milk
yield and total protein concentration. Whey protein and -casein proportions of total protein were
significantly reduced, casein/total protein ratio was elevated and rennet coagulation properties were
partly impaired by the alpine hay, whereas plasmin and plasminogen-derived activity were not
clearly affected.
Key words: altitude, forage quality, milk protein composition, plasmin, cheese-making
Introduction
Milk production on alpine sites is one form of using forage resources in less favoured areas, often
combined with the production of an unconventional cheese of high quality. However, high altitude
and insufficient forage quality may impair milk protein synthesis and composition, with the
consequence of undesired side-effects on cheese yield and quality. Although genetically limited to a
small variation, casein and its fractions can be affected by environmental factors such as season and
ambient temperature (Kroeker et al., 1985; Lacroix et al., 1994; Reichardt et al., 1995). Particularly,
a deficit in energy supply, as it is common on high altitude pastures, might be detrimental for
protein synthesis and even casein proportion of total milk protein (Reichardt et al., 1995). A
decreased casein proportion leads to lower cheese yield (Melilli et al., 2002) and may also modify
the processes of coagulation and cheese ripening. The aim of this experiment was to study
independently of each other the influences of high altitude and alpine origin of the forage on milk
protein content and composition, proteolytic activity and rennet coagulation properties of milk.
Materials and Methods
With a two-factorial design, the effects of altitude and of forage quality were tested simultaneously.
Four groups of 3 lactating Holstein and 3 Brown Swiss cows each were investigated in the
experiment. The groups were balanced according to stage of lactation, energy-corrected milk yield,
protein content and -casein genotype of the cows. Two groups were fed with hay ad libitum, one
of them (G1) at ETH research station 'Weissenstein' at 2000 m a.s.l. and the second (G2) at ETH
research station 'Chamau' at 400 m a.s.l. A third group (G3) was pairfed to G1 and kept at 'Chamau'
for evaluating the impact of a decreased food intake, which may occur under alpine conditions
(Christen et al., 1996). All these groups were fed with hay alone either from alpine (AH) or from
lowland origin (LH) following a change-over design. After one week of adaptation to the hay diets
the hays were fed to half of the cows within each group in the sequence alpine-lowland-alpine, and
in the sequence lowland-alpine-lowland to the other half. Each period within sequence lasted for 21
days. Thus the average forage quality for each group was equal and the different forages were tested
under both, alpine and lowland conditions. The alpine hay had higher fiber and lower crude protein
contents and a clearly lower digestibility compared to the lowland hay. Consequently, the net
energy supply from the alpine hay was reduced from 5.8 to 5.1 MJ NEL/kg DM. A fourth group
(SR) was fed with a silage ration and concentrates corresponding to milk yield.
All cows were tethered in barns. In the last week of each 21-day period, milk was sampled at
every milking. Cows were weighed and blood samples were taken twice in every sampling week.
All individual milk samples were analysed for major milk constituents, including protein content,
with infrared technique using a Milkoscan 4000 (Foss Electric, Hillerød, Denmark). Over the whole
week, aliquot milk samples were pooled for each animal. Proportions of milk protein fractions were
quantified with RP-HPLC (Merck-Hitachi, Darmstadt, Germany) on a C4 column (Vydac,
Hesperia, USA), adapting the method of Bordin et al. (2001). Plasmin and plasminogen-derived
activity were measured modifying the method of Richardson & Pearce (1981) on a microplate
fluorescence reader (Tecan, Männedorf, Switzerland). Rennet clotting properties were evaluated
with a Lattodinamografo (Foss, Padua, Italy) after skimming and adding chymosin (Maxiren, GistBrocades, Seclin Cedex, France). Rennet clotting time (RCT) is the time (min) from adding
chymosin until coagulation begins. K20 is a measure of the dynamics of coagulation and expresses
the time (min) from the start of the coagulation until a defined curd firmness is reached. Plasma
metabolites were measured with commercial photometric test kits (for -hydroxybutyrate from
Sigma, Buchs, Switzerland; for glucose from Roche, Basle, Switzerland).
Statistical analyses were done using the procedure ‘Mixed’ of SAS with two models. The
first included forage, group, breed, protein genotypes and period as fixed and animal within breed ×
group as random effect. All interactions were considered. Initial values obtained two weeks prior to
the experiment were used as a covariate. This model was applied on the groups G1-G3. For
inclusion of SR, a second model considering ‘diet type’ instead of hay and group was applied. In
Figure 1 only the values for SR were derived with this model.
Results and Discussion
The difference between G1 and G2 gave the best estimate of the altitude effect because forage
intakes were similar between these ad libitum-fed groups. Only in the initial week of the whole
experiment, G1 showed severe intake refusals, which were mirrored in the pairfed G3 cows. In both
groups this led to a persistent negative energy balance, indicated by elevated plasma hydroxybutyrate, reduced glucose levels and by slight body weight losses. Consequently, results
where G1 and G3 are similar but different from G2 are interpreted as to have been influenced more
likely by the initial undernutrition than directly by the altitude.
Altitude did not cause a reduced milk yield (Fig. 1). Milk protein content declined in both G1
(P<0.05) and G3 and therefore an effect of the initial undernutrition is assumed. Correspondingly
the casein content of milk was lower in G1 and G3 than in G2 (P<0.05). Casein/total protein
proportion was lower in the alpine group (G1) than in the lowland hay-alone groups. Within the
casein fraction, the -casein was lower in the milk of the alpine group; all other casein fractions
were rarely affected by altitude. Plasminogen derived activity of the alpine group was decreased
(P<0.1), but this was not accompanied by alterations of the plasmin activity. A clear negative effect
of high altitude on the renneting properties was found, which is expressed in prolonged rennet
clotting time (P<0.05) and a trend towards retarded dynamics (K20).
Daily energy corrected milk yield
*
Milk protein content
36
g / kg milk
kg ecm / day
30
25
a
20
b
15
Casein concentration in skim milk
g / kg skim milk
32
a
ab
a
30
b
AH LH SR
Whey protein concentration in skim milk
*
b
b
G1 G2 G3
26
24
8
0
b
a
6
4
0
AH LH SR
Casein proportion of total protein
82
AH LH SR
16
a
81
G1 G2 G3
-casein proportion of total casein
b
80
79
g/100 g casein
G1 G2 G3
g/100 g protein
b
30
AH LH SR
g/kg skimm milk
G1 G2 G3
a
b
14
12
0
0
G1 G2 G3
G1 G2 G3
AH LH SR
-casein proportion of total casein
AH LH SR
Plasminogen-derived activity of milk
38
1.2
36
mg/l
g/100 g casein
32
0
0
28
a
34
1.0
34
0.8
0.0
0
G1 G2 G3
AH LH SR
Rennet clotting time
18
G1 G2 G3
AH LH SR
Coagulation dynamics (K20)
a
6
b
b
min
min
15
12
4
9
2
0
0
G1 G2 G3
AH LH SR
G1 G2 G3
AH LH SR
Figure 1. Influence of group treatment and diet type on milk yield and composition. Group
treatments are: G1: alpine site, ad libitum; G2: lowland site ad libitum; G3: lowland site, pairfed
to G1. Diet types are: AH: alpine hay; LH: lowland hay; SR: silage ration with concentrates. Bars
carrying different letters are significantly different within the group or the hay factor at P>0.05.
Asteriscs above the SR bars indicate significant differences between the silage ration and the two
hay types.
Both hays were not sufficient in quality to cover the energy and protein requirements of the
lactating cows, and consequently the milk yield of the groups G1-G3 was drastically lower for both
hays than in the group, which was constantly fed on silages and concentrate (SR). The hay with
alpine origin further depressed milk yield compared to the lowland hay. Additionally the milk
protein content was reduced (P<0.001) by use of the alpine instead of the lowland hay on average of
both altitudes. This decrease in milk protein content was relatively more pronounced in the whey
proteins (P<0.01) than in the caseins, which resulted in a higher (P<0.01) casein/total protein ratio
of the milk produced from the alpine hay. Within the caseins, -casein was significantly reduced
(P<0.05), whereas -casein was slightly increased when comparing the alpine hay with the lowland
hay. On the basis of the Van-Slyke formula (Melilli et al., 2002), the reduced milk protein
concentration which is caused by the alpine hay type would lead to a reduction of cheese yield of
260 g/100 kg milk when the casein/total protein ratio would be constant. Considering the higher
casein proportion with the alpine hay in the formula, the reduction is only 205 g/100 kg milk.
Casein content of milk was positively affected (P<0.05) by the silage ration. For the other
traits related to milk protein quality, silage ration was never different from lowland hay and one
factor which may have led to the observed changes was probably the particularly low supply of
nutrients from the alpine hay. Similar to altitude, the type of feed did not influence the plasmin
activity of milk, but the plasminogen-derived activity was elevated by the use of any hay compared
to the silage ration, (P<0.1 for the lowland hay type). The rennet clotting time was not affected by
the diet type, whereas the dynamics of coagulation was weaker with the hays, especially with the
alpine hay, compared to the silage ration.
This study shows, that, although small, significant changes in the protein composition can be
evoked by the diet. The extent of the difference of 1% in the casein proportion caused by changing
the hay type is more or less equal to the phenotypical difference which occurs between the -casein
AA and AB genotypes in several studies (Reichardt et al., 1995) and also was found in our study.
As shown, these alterations have a relatively small impact on the cheese yield. The higher casein
proportion caused by the alpine hay did not lead to improved rennet coagulation properties, this
probably because of the concomitantly reduced milk protein content. Although altitude had no
significant influence on milk protein content and composition, the significantly delayed rennet
clotting time seems to reflect the adverse trends in protein concentration and casein proportion.
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