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273
Original article
Abstract Teleme cheese was made from ovine, caprine and bovine milk with thermophilic (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus), mesophilic (Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris) and mixed thermophilic-mesophilic (1:1) cultures. Nine lots of cheese each time (five repetitions) were made in order to study the proteolysis of
cheeses during ripening and storage up to 6 months. All cheeses showed similar profiles of proteolysis. However, the rate of proteolysis was different. The thermophilic and the mesophilic cultures
resulted in cheeses with the highest and the lowest degree of proteolysis, respectively. Proteolysis
was of a descending order in cheeses made from sheeps, goats and cows milk when estimated on
the basis of nitrogenous fractions (total nitrogen, water soluble nitrogen, nitrogen soluble in 5%
phosphotungstic acid and nitrogen soluble in 12% trichloroacetic acid), whereas it was highest in
cows milk cheeses and lowest in goats milk cheeses when estimated on the basis of electrophoresis
and RP-HPLC. Multivariate analysis of the data revealed that cheeses could be more correctly clustered by the type of milk or culture than by the stage of ripening.
Teleme cheese / white-brined cheese / proteolysis / starter culture
- Teleme)
(Teleme cheese),
(Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus)
(Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris) (1:1) 3
9 5
6
5%
12%
/ / /
* Corresponding author (): instgala@otenet.gr
274
Rsum Influence du type de lait et de levain sur la protolyse de fromage Tlm au cours
de laffinage. Du fromage Tlm a t fabriqu avec du lait de brebis, du lait de chvre et du lait
de vache en utilisant des levains lactiques thermophiles (Streptococcus thermophilus, Lactobacillus
delbrueckii subsp. bulgaricus), msophiles (Lactococcus lactis subsp. lactis, Lactococcus lactis
subsp. cremoris) ou mixtes thermophiles-msophiles (1 : 1). Neuf fabrications (cinq rptitions) ont
t ralises pour tudier la protolyse des fromages au cours de laffinage pendant 6 mois. Tous les
fromages avaient un profil protolytique similaire, mais le degr de protolyse tait diffrent. Les
fromages fabriqus avec des levains thermophiles taient les plus protolyss et ceux fabriqus avec
des levains msophiles les moins protolyss. La protolyse observe, estime par les fractions azotes (azote total, azote soluble en phase aqueuse, azote soluble dans le PTA 5 % et azote soluble
dans le TCA 12 %), diminuait dans lordre lait de brebis, lait de chvre et lait de vache mais elle
tait la plus forte pour le fromage prpar avec du lait de vache et la plus faible pour le fromage
prpar avec du lait de chvre quand elle tait estime par lectrophorse et HPLC phase inverse.
Lapplication de lanalyse multivarie a rvl que la classification des fromages tait plus correcte
avec le type de levains lactiques ou de lait utiliss quavec le degr de maturation.
Tlm / fromage en saumure / protolyse / levain / maturation
1. INTRODUCTION
Proteolysis is an important biochemical
process in the ripening of most cheese varieties. Proteolysis, which contributes to the
development of cheese texture and flavor,
is due to the action of enzymes from (a)
coagulant, (b) milk (general plasmin), (c)
starter bacteria, (d) the adventitious nonstarter microflora and (e) secondary starter
(in some cheese varieties). The proteolytic
system of lactic acid bacteria (LAB) consists of proteinases, which hydrolyze
caseins, and large peptides and peptidases,
which are responsible for the liberation of
small peptides and free amino acids (FAA),
an essential step in cheese ripening [43].
White-brined cheeses, in which Teleme
cheese is included, form a special category
of cheeses. Their main characteristic is that
they are ripened and stored in brine. Traditionally, Teleme cheese was manufactured
from non-pasteurized milk, but nowadays it
is manufactured on an industrial scale. Rigorous control of the production and maturation process is necessary. The selection of
the starter culture is one of the most important steps in the manufacture of high-quality cheeses. In traditional Teleme cheese
manufacture, yogurt is used as a starter. A
mixture of commercial starters, including
mesophilic starters, are used in industrial
production [2]. Studies concerning the
effect of the starter cultures used on the ripening of Teleme cheeses have not been previously reported in a systematic way [20,
bulgaricus and Streptococcus thermophilus - the ratio of thermophilic to mesophilic microorganisms was 1:1, and (iii) a
freeze-dried concentrated mesophilic culture (M), R-703 (Hansens Laboratorium,
Denmark) consisting of the microorganisms Lactococcus lactis subsp. lactis and
Lactococcus lactis subsp. cremoris. For
one week, on a day after day basis, one
batch of cheese-making of sheeps, goats
or cows milk took place. On the day of the
cheese-making, the milk (sheeps, goats or
cows) was divided into three equal parts
and in each part one of the three cultures
was used. Therefore nine lots of cheese
were obtained in one week. This procedure
was repeated during five subsequent weeks.
The reported results are the averages of five
trials.
2.2. Cheese-making and sampling
Teleme cheese was manufactured as
described previously [33]. Briefly, the milk
was standardized to casein to fat ratios of
0.75, 0.64 and 0.73 for the sheeps, goats
and cows milk, respectively, to obtain a
first quality cheese that was 56% moisture
and 43% fat in dry matter [15]. The standardized milk was pasteurized at 63 C for
30 min and then cooled at 37 C. After
cooling, one of the cultures mentioned
already was added to it, at a quantity of
0.5% (v/v). To assist curdling of the milk
CaCl2 solution (40%, w/v) was added. Rennet
powder HA-LA (Hansens Laboratorium,
Horsholm, Denmark) was added at a quantity
of 3.0 g per 100 kg milk, at 33 C. Drainage
was under pressure and salting took place
in brine (14% NaCl, w/w) for 16h at 19 C.
After manufacture, cheeses were ripened in a warm room (19C), until pH and
moisture values reached the limits of 4.6
and 56%, respectively, and afterwards
cheeses were transferred to a cold room
(5 C) for up to 6 months (period of sampling). The relative humidity of the rooms
was medium to avoid rusting of the tins,
since Teleme cheese is ripened and stored
in sealed tins. Teleme cheeses made from
sheeps milk using thermophilic, thermophilic-mesophilic or mesophilic culture
were transferred to the cold room at 24, 16
275
276
2.432ab
2.47a
2.392b
2.392a
2.552b
2.36a
2.3812
2.381
2.3612
2.362a
2.452b
2.302a
2.531a
2.66a
2.301b
2.261a
2.461b
2.32c
2.351
2.3812
2.291
2.221ab
2.371a
2.161b
S
G
C
S
G
C
S
G
C
S
G
C
R(ii)
60
120
180
2.422a
2.4212a
2.302b
2.482a
2.462ab
2.382b
2.442a
2.5012a
2.29b
2.452a
2.53b
2.291c
2.4212a
2.50b
2.3012c
19.021a
13.001b
17.101c
17.191a
11.711b
11.751b
16.081a
10.191b
10.11b
13.831a
9.921b
10.411b
8.751a
6.991b
7.151c
12.542a
9.192b
10.282c
12.492a
9.372b
9.682b
10.192a
8.242b
9.66c
6.532a
8.272b
8.172b
4.602a
5.172b
5.202b
MX
WSN, %TN
11.742a
9.002b
10.152c
11.282a
7.763b
9.042c
11.052a
7.412b
9.06c
5.873a
7.622b
8.572c
3.103a
3.693b
4.993c
2.691
2.861
2.941
2.041
1.991
2.201
2.141
2.111
2.001
2.091
2.041
1.991
1.721a
1.601b
1.361c
2.3212ab
2.062a
2.422b
1.991a
1.762b
1.902ab
1.932a
1.682b
1.991a
1.622
1.532
1.662
1.302a
1.102b
1.182c
MX
PTA-N, %TN
2.142ab
1.952a
2.282b
1.822a
1.572b
1.872a
1.763
1.622
1.812
1.921a
1.392b
1.682c
1.123
1.102
1.103
11.921a
10.321b
10.311b
8.74ab
10.051a
7.891b
8.181a
8.431a
7.04b
7.321
6.951
6.901
3.101a
2.801b
2.901c
(i) Means of five cheese-making trials. (ii) Day cheeses were transferred to cold room (see text for details).
a,b,c Means of each parameter in the same column of the same day with different letters are significantly different (P < 0.05).
1,2,3 Means of each parameter in the same raw of the same day with different numbers are significantly different (P < 0.05).
2.402a
2.49b
2.352a
2.471a
2.51a
2.281b
S
G
C
MX
Age
Kind
(days) of milk
9.112a
6.712b
7.532b
8.68a
6.442b
6.402b
7.562a
6.292b
6.87ab
7.111a
5.612b
5.682b
2.812a
1.992b
2.002b
MX
TCA-N, %TN
8.512a
6.352b
7.312c
8.07a
5.902b
6.052b
6.883a
5.812b
6.73a
5.042a
5.492ab
5.702b
2.143a
1.803b
1.903c
Table I. Changes of nitrogenous fractions of Teleme cheese made from sheeps (S), goats (G) or cows (C) milk using a thermophilic (T),
a mesophilic (M) or a mixture of thermophilic and mesophilic (MX) starter during ripening and storage(i).
278
Figure 1. Electrophoregrams of Teleme cheese made with sheeps, goats or cows milk with the
mixed (thermophilic-mesophilic) culture during ripening and storage. Lanes 12: total sheeps
casein; 34: 1-d cheese; 56: when cheese was transferred to cold room (see text for details); 78:
60-d cheese; 910: 120-d cheese; 1112: 180-d cheese.
279
Figure 2. Electrophoregrams of the WSE of Teleme cheese made with sheeps, goats or cows
milk with the mixed (thermophilic-mesophilic) culture during ripening and storage. Lanes 12: 1-d
cheese; 34: when cheese was transferred to cold room (see text for details); 56: 60-d cheese; 78:
120-d cheese; 910: 180-d cheese.
90.62a
94.5b
84.7c
86.52a
89.7a
76.81b
82.72a
84.7a
74.212b
87.91a
93.2b
83.7c
86.41a
86.2a
75.11b
75.91a
84.2b
70.21c
S
G
C
S
G
C
S
G
C
60
120
180
84.13ab
85.4a
77.62b
91.33a
89.7a
82.72b
92.53a
94.5b
85.3c
96.72
98.6
94.02
54.41a
73.4b
34.41c
57.31a
83.5b
47.71c
58.11a
85.7b
49.21c
72.11a
91.0b
55.71c
59.92a
79.0b
50.32a
64.92a
84.9b
52.71c
75.02a
86.2b
54.41c
83.712a
91.5b
73.42c
63.82a
79.1b
61.73a
71.43a
85.0b
64.42a
78.93a
89.0b
72.92c
87.52ab
93.9a
77.82b
37.41a
65.21b
32.71c
43.71a
67.51b
37.91a
54.21a
73.7b
42.21c
61.31a
83.9b
40.81c
(i) Means of five cheese-making trials. (ii) Day cheeses were transferred to cold room (see text for details).
a,b,c Means of each parameter in the same column of the same age with different letters are significantly different (P < 0.05).
1,2,3 Means of each parameter in the same row of the same age with different numbers are significantly different (P < 0.05).
94.72a
95.0a
91.42b
90.01ab
95.0a
86.11b
S
G
C
R(ii)
55.02a
69.612b
43.02c
58.52a
71.312b
45.02c
60.62a
77.3b
47.212c
66.812a
85.7b
57.22c
59.33a
73.52b
44.13c
62.32a
75.42b
45.22c
67.53a
81.0b
47.92c
70.72a
88.9b
58.82c
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
MX
MX
MX
s-casein (%)
-casein (%)
S
G
C
Kind of
milk
Age
(days)
Table II. Changes in residual -casein (%), s-casein (%) and in water-soluble nitrogen (WSN, %), in polyacrylamide gel, of Teleme cheese made
from sheeps (S), goats (G) or cows (C) milk using a thermophilic (T), a mesophilic (M) or a mixture of thermophilic and mesophilic (MX) starter
during ripening and storage(i).
280
E.C. Pappa et al.
281
61.172a
55.442b
66.862c
66.432a
63.882a
69.802b
68.332a
66.442b
70.692c
70.332a
68.082b
72.532c
68.611a
65.541a
73.241b
73.121ab
70.891a
75.351b
75.671a
73.441a
78.691b
76.111a
75.251a
79.161b
S
G
C
S
G
C
S
G
C
S
G
C
R(ii)
60
120
180
65.593a
63.673b
68.813c
63.343a
60.663b
66.183c
61.623a
56.713b
64.543c
58.732a
50.483b
58.053a
33.243a
40.743b
51.25c
23.891a
24.751a
20.841b
24.331a
26.551a
21.351b
26.881ab
29.111a
24.651b
31.391a
34.961a
26.761b
51.491a
50.741a
46.46b
29.672a
31.892b
27.472c
31.672a
33.562b
29.312c
33.572a
36.122b
30.202c
38.832a
44.562b
32.482c
60.432a
55.922b
47.8C
MX
HB(%TA)(iii)
34.413a
36.333b
31.193c
36.663a
39.343b
33.823c
38.383a
43.293b
35.463c
41.272a
49.523b
41.953a
66.763a
59.263b
48.75c
0.311a
0.331a
0.261b
0.321a
0.361a
0.271b
0.371ab
0.411a
0.331b
0.461a
0.531a
0.371b
1.041a
1.031a
0.87b
(i) Means of three cheese-making trials. (ii) Day cheeses were transferred to cold room (see text for details).
(iii) Results are expressed as % of the total area (TA) of the chromatogram.
a,b,c Means of each parameter in the same column of the same age with different letters are significantly different (P < 0.05).
1,2,3 Means of each parameter in the same row of the same age with different numbers are significantly different (P < 0.05).
39.572a
44.082b
52.2C
48.511a
49.261a
53.54b
MX
HL (%TA)(iii)
S
G
C
Kind
of milk
Age
(days)
0.422a
0.472b
0.382c
0.462a
0.512b
0.412c
0.512a
0.572a
0.432b
0.632a
0.802b
0.492c
1.532a
1.272b
0.92c
MX
HB/HL
0.533a
0.573b
0.453c
0.583a
0.653b
0.513c
0.623a
0.763b
0.553c
0.702a
0.983b
0.723a
2.003a
1.453b
0.95c
Table III. Changes in the area of hydrophilic (HL) and hydrophobic (HB) peptides (%TA) and the ratio of HB/HL of the water-soluble fraction of
Teleme cheese made from sheeps (S), goats (G) or cows (C) milk using a thermophilic (T), a mesophilic (M) or a mixture of thermophilic and
mesophilic (MX) starter during ripening and storage(i).
282
E.C. Pappa et al.
283
Figure 3. RP-HPLC chromatograms of the WSE of Teleme cheese with sheeps, goats or cows
milk with the mixed (thermophilic-mesophilic) culture at 1 and 180 days.
were observed on any sampling day, suggesting that the mode of casein breakdown
was similar in all cheeses. It can be seen that
there were peptides produced with faster
electrophoretic mobility (Rf) than scaseins, with mobility slower than -casein
and with mobility between s- and caseins. Abd El-Salam and Alichanidis [1]
found that the hydrolysis products in whitebrined cheeses with Rf faster than s-CN
are produced from the action of chymosin
on s1-CN, whereas the products with
284
Table IV. Spearman correlation coefficients between the different variables investigated(i).
WSN
PTA
TCA
TN
HL
HB
HB/HL
s-CN
-CN
WSN
PTA
0.88**
TCA
0.93**
0.88**
TN
0.38*
0.40**
0.35*
HL
0.85**
0.88**
0.85**
0.53**
HB
0.85** 0.88**
0.85**
0.53**
0.99**
HB-HL
0.85** 0.88**
0.85**
0.53**
0.99**
0.99**
s-CN
0.79** 0.80**
0.75**
0.58**
0.87**
0.87**
0.87**
-CN
0.75** 0.83**
0.75**
0.53**
0.86**
0.86**
0.86**
0.91**
0.74** 0.76**
0.72**
0.63**
0.85**
0.85**
0.85**
0.98**
Total area
of the WSN
0.88**
in PAGE
(i)Abbreviations of the variables are explained into the text.
*P < 0.05; **P < 0.01.
285
Figure 5. Linear discriminant functions (D1 and D2) for cheese samples at 1, 60, 120, 180 days of
ripening time as well as the day cheeses entered the cold room (R). Both D1 and D2 were significant
(P < 0.001).
286
Some overlapping between R, 60- and 120day-old samples was observed, which
caused the misclassification. As ripening is
a continuous process, the groups may not be
completely separated. The most important
predictor variables in classifying samples
into the correct group were TN, TCA, HL
and the total area of the WSN in PAGE.
4.2. Effect of type of culture
Analysis of the TN and soluble N, as estimated with the Kjeldahl method (Tab. I),
showed that the lowest TN values of mature
cheeses made with the thermophilic culture
were due to the fact that this culture was the
most proteolytic, and therefore the loss of
water-soluble nitrogenous compounds in
the brine was higher than the other two cultures. Cheeses made with the thermophilic
and the mesophilic cultures showed the
highest and the lowest % WSN/TN values,
respectively. This is in agreement with the
results of other studies for white-brined
cheese made with cultures similar to those
of this study [20, 32]. Thermophilic culture
resulted in cheeses with higher PTA-N
content than the cheeses made with the
other two cultures. Generally, the latter did
not differ significantly in the PTA-N content (P > 0.05). Similar results were also
found by Gomez and Malcata [12]. The %
TCA/TN values of cheeses made with the
thermophilic culture were significantly higher
(P < 0.05) than those of cheeses made with
the other two cultures. This is in agreement
with the results found by Pappa and Anifantakis [32] and Gomez and Malcata [12].
From this study, it is observed that the thermophilic and the mesophilic cultures
resulted in cheeses with the highest and the
lowest (P < 0.05) production of HL peptides, respectively (Tab. III). This is in
agreement with the results of Steffen et al.
[40] and Garde et al. [9].
Thermophilic culture showed the highest
hydrolysis of s- and -CN, and mesophilic
the lowest when cheeses were made from
sheeps or cows milk, as estimated with
urea-PAGE (Tab. II). This is in agreement
with the results reported by others [4, 9, 13,
44]. However, the three cultures showed the
same rate of proteolysis (P > 0.05), in rip-
287
Figure 6. Linear discriminant functions (D1 and D2) for cheese samples made using a thermophilic
(T), a mesophilic (M) and a mixture of thermophilic-mesophilic (MX) culture. D1 was significant
(P < 0.001) but D2 was not significant (P > 0.05).
288
Figure 7. Linear discriminant functions (D1 and D2) for cheese samples made from sheeps, goats
and cows milk. Both D1 and D2 were significant (P < 0.001).
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