E/-Sada, Cheese ripening
spreadability in the case of heat step ripening, as here
only the continuous fat phase which provides the ,lubri­
cation" appears to increase. We wish to answer this
question in our next work.
5. References
F., DEMAN, J.M.: J. Dairy Sci. 47
(1)32-36 (1964)
DEROANNE, C.: Le Lait 54 39-55 (1976)
KNOOP, A.M.: Kieler Milchw. Forschgsber. 15 (4)
319-325 (1963)
KNOOP, E.: Milchwissenschaft19(4) 168-172(1964)
PRECHT, D., PETERS, K.H.: Milchwissenschaft 36
(10) 616-620 (1981)
PRECHT, D., PETERS, K.H.: Milchwissenschaft 36
{11) 673-676 (1981)
SHERBON, J.W., DOLBY, R.M.: J. Dairy Res. 39
319-324(1971)
SHUKLA, A., BHASKAR, A.R., RIZVI, S.S.H., MUL­
VANEY,S.J.:J. Dairy Sci. 7745-54(1994)
SCHAFFER, B., SZAKALY, S.: Mifchwissenschaft
43(9)557-560(1988)
SZAKALY, S., SCHAFFER, B.: Milchwissenschaft
43(9)561-564(1988)
TIMMS, R.E:Austr.J. DairyTechnol.3547-53(1980)
(1) CANTABRANA,
(2) (3) (4) (5) (6) (7) (8) (9) (10)
(11)
6. Summary
SCHAFFER, B., SZAKALY, S., LORINCZV, D., BElAGYI,
J.: Structure of butter Ill. Effect of modification of cream
ripening and fatty acid composition on the melting
properties of butter fat. Milchwissenschaft 54 (2) 82-85
(1999).
45 Butter structure (melting properties)
By the differential scanning calorimetric method the
melting and crystallization characteristics of fat of the butt­
ers made of cream ripened traditionally (.summer-type")
and by the heat step method (.winter-type") and not en­
riched and enriched with a low melting point (LMP) milk fat
fraction can be well detected.
It has been stated that due to the heat-step ripening,
compared to the traditional ripening method, the single
melting peak registered in the temperature range of Q-20°C
breaks down into 2 peaks. The addition of a LMP milk fat
85
fraction decreases the temperature of the melting peak of
butter made of traditionally ripened cream. However, the
temperature of the double peak of butter made of cream rip­
ened by the heat step method does not change, but the en­
thalpy ofthe lower temperature melting peak will be higher.
The results correspond to the knowledge on the ho­
mogenous and particle structure of butter. The melting
point of fat of butter of homogenous structure made by the
traditional cream ripening decreases due to the addition of
a LMP fraction. At the same time, the LMP fraction will be
incorporated in the continuous fat phase surrounding the
particles in butter of mainly particle structure made of
cream ripened by the heat step method.
SCHAFFER, B., SZAKALY, S., LORINCZV, D., BElAGYI,
J.: Struktur von Butter.lll. EinfluB der VerDnderung von
Sahnereifung und Fettsaurenzusammensetzung auf die
Schmelzeigenschaften von Butterfett. Milchwissenschaft
54 (2) 82-85 (1999).
45 Butterstruktur (Schmelzeigenschaften)
Durch das kalorimetrische Differentiai-Scan-Verfahren
konnen die Fettschmelz- und Kristallisierungseigen­
schaften von Butter, hergestellt aus traditional! gereifter
Sahne (Sommertyp) bzw. nach dem .Hitzestufenverfah­
ren" (Wintertyp), bei Anreicherung bzw. Nichtanreiche­
rung mit einer Milchfettfraktion mit niedrigem Schmelz­
punkt (LMP) gut festgestellt werden.
Es zeigte sich, daB sich durch die Hitzestufenreifung ­
verglichen mit dem traditionellen Reifungsverfahren- der
einzelne Schmelzpeak im Temperaturbereich 0-20°C in
zwei Peaks darstellt. Der Zusatz einer LMP-Milchfettfrak­
tion senkt die Temperatur des Schmelzpunktes von Butter
aus traditionell gereifter Sahne. Jedoch verandert sich die
Temperatur des Doppelpeak von Butter aus mit der Hit­
zestufenmethode gereifter Sahne nicht, wahl aber ist die
Enthalpie des niedrigeren Schmelzpunktes hoher.
Die Ergebnisse entsprechen der Kenntnis iiber die ho­
mogene und Partikei-Struktur von Butter. Der Schmelz­
punkt von Butterfett mit homogener Struktur aus traditio­
nailer Sahnereifung sinkt durch das Zufiigen einer LMP­
Fraktion. Gleichzeitig wird die LMP-Fraktion in die konti­
nuierliche Fettphase integriert, die die Partikel in Butter mit
vorrangiger Partikelstruktur bei mit dem Hitzestufenver­
fahrens gereifter Sahne umgibt.
Evaluation of commercial adjuncts for use in cheese ripening:
1. Enzymatic activities and autolytic properties of freeze-shocked
adjuncts in buffer system
By M ..EL-SODA1, S.A MADKOR2 and P.S. TONG3
1
Department of Dairy Technology, Faculty of Agriculture, University of Alexandria, Alexandria, Egypt
Department of Dairy Technology, Faculty of Agriculture, Minia University, EI-Minia, Egypt
3
DairY Products Technology Center, California Polytechnic State University, San Luis Obispo, CA 93407
2
1. Introduction
The ripening of many cheeses can be a slow and
complex process that is not fully controllable. Many
cheeses do not develop the desired quality attributes
despite extended aging times. The major biochemical
Milchwissenschaft 54 (2) 1999
changes during cheese ripening involve breakdown of
carbohydrates, fat and proteins by rennet, milk indige­
nous enzymes, starter bacteria and secondary micro­
floraofcheeseandtheirrelatedenzymes (15, 16). These
EI-Soda, Cheese ripening
86
cells, MRS broth was inoculated with an active culture
changes provide the characteristic flavor and texture
of the organisms and sub-cultured in the same medium.
profiles to ripened cheeses. Most of the approaches to
At early stationary phase, cells were harvested by cen­
cheese ripening enhancement fall into 1 of 4 categories:
trifugation at 1500 g for 30 min at 4
elevated ripening temperature, enzyme addition to milk
The cell pellet
was washed twice with 0.01 potassium phosphate
or curd, addition of slurries containing cheese flavor
components and addition of modified or non-modified
buffer, pH 7.0 and resuspended in the same buffer to
obtain a total viable cell concentration in the range of
cheese related microorganisms (14, 17, 33).
108-10 12 cfu/ml.
The observation that raw milk cheese develops more
intense flavor than pasteurized milk cheese has stimu­
2.2 Freeze-shocking of the cell cultures
lated interest in adding non-starter lactic acid bacteria
as adjunct cultures to pasteurized milk used in cheese
The cell suspension was frozen at -20°C for 24h,
manufacture (25, 29, 31 ). The addition of defined strains
thawed in a water bath at 40
and used immediately
of lactobacilli as adjuncts to Lactococcus starter cultures
after thawing.
promotes cheese ripening (25, 31 ). Lactobacilli can grow
and multiply under selective conditions of Cheddar
2.3 Preparation of the cell free extract (CFE)
cheese ripening, while other bacteria decrease in num­
The methods of EI-SODA and DESMAZEAUD (10)
ber (28, 31 ). However, addition of moderate amount of
were used for the preparation of the CFE with some
3
7
an adjunct culture (10 -10 /ml) could induce an abnor­
mally rapid lactic fermentation and excess acid produc- · modifications. A mixture of 2 parts of alumina powder
(Type A-5, Sigma Chemical Co., St. Louis, MO, USA) to
tion. Several methods are described in the literature to
1 part of cell pellet were ground with a mortar and pes­
minimize the rate of lactic acid production of the adjunct
tle. The CFE were stored frozen at -20
and carefully
cultures before inoculation in the cheese milk. These
thawed before use.
include: lysozyme treated cells, solvent treated cells,
neutralized inactivated cultures, Lac- mutants, heat,
2.4 Cellular enzyme assays
freeze or spray dried shocked cells (14, 17). Freeze­
Aminopeptidase and dipeptidyl-aminopeptidase ac­
shocking at sub-optimal temperature to damage the cell
tivities of the CFE were measured using synt~etic ,:r
wall and membrane causes a partial loss of the cell's
nitroanilides derivatives of amino acids and dipeptides
ability to produce acid (22, 23). Freezing temperature
as described by El ABBOUD! eta/. (9). The esterolytic
varied from -20 to -30
for 20 to 36 h (11, 14). Also,
activities were studied using ,:rnitrophenyl and o-nitro­
freeze-shocking induced cell lysis with only a limited
reduction of cellular proteinase, peptidase and esterase . phenyl derivatives of fatty acids according to the
method of BRANDL and ZIZER (4). One unit of enzyme
activities (11, 18, 22, 23).
activity was defined as an increase in 0.01 absorb­
Death and lysis of the adjunct cells can release intra­
ance/min at 410 nm. The specific activity was ex­
cellular enzymes into the cheese matrix where they play
pressed as the number of activity units/mg of protein of
a role in protein and fat hydrolysis. OHMIYA and SATO
the CFE. The method of LOWRY et a/. (27) was used
(30) studied the conditions to promote autolysis in lac­
for determination of the protein content of the CFE.
tobacilli, and they demonstrated that autolysis was en­
hanced by freezing at -20 °C. It was reported that lacto­
2.5 Measurement of the rate of autolysis and bacilli are highly susceptible to cryo-injury, but it seems
enzyme release that their aminopeptidase activities are quite resistant to
freezing (23). Reports on peptide hydrolyzing enzymes
A portion of cell suspension (-10%, v/v) was added to
and autolytic properties of freeze-shocked lactobacilli
0.01 phosphate buffer (pH 5.2) containing 0.5 M sodium
(21, 31) suggested that enhanced cheese flavor ob­
chloride to obtain an optical density of 0.8 to 1.0 at 650 nm
tained with added lactobacilli can be partly attributed to
and incubated at 30°C. After set time intervals (0, 3, 6,
cell lysis and their associated high enzyme activities.
24 and 48 h) the percentage decrease in optical density
Starter manufacturers are now offering commercial
was measured and expressed as% autolysis (32).
adjunct cultures to improve and accelerate cheese flavor
For determination of enzyme release, aliquots of the
development. However, little scientific information is avail­
autolysing cell suspension were collected at the same
able on the properties needed for successful adjunct in
intervals mentioned above and centrifuged at 1500 g for
cheese such as: the level of peptidase and esterase
30 min at 4
The resultant supernatant containing the
activities, rate of autolysis and intracellular enzyme re­
intracellular enzymes were stored at -20°C, then as­
lease. The objective of the present work was to screen
sayed for enzyme activities. The extent of enzyme activ­
several commercial adjunct cultures by quantifying their
ity release was calculated as follows:
enzyme activities and autolytic properties in buffer system.
% Enyzme activity release=
oc.
oc
oc
oc
oc.
2. Materials and methods
2. 1 Cultures and cell cultivation
Eight Lb. helveticus and 7 Lb. casei were evaluated
during this work. Fourteen of these cultures were chosen
because they have been recommended for use as ad­
. juncts by commercial culture suppliers.· A non-com­
mercial strain of Lb. helveticus culture obtained from
Oregon State University (Bill Sandine, personal com­
munication) was also evaluated. For the cultivation of
enzyme activity measured in the supernatant x
total enzyme activity of the CFE
100
3. Results and discussion
3. 1 Specific enzyme activities in cell free extract ..
o(Lactobacillus strains
Results of enzyme activities in CFE of different
strains of lactobacilli are given in Table 1. Aminopepti­
dase and dipeptidyl-aminopeptidase specific activities
Milchwissenschaft 54 (2) 1999
E/-Soda, Cheese ripening
87
Table 1: Aminopeptidase, dipeptidylaminopeptidase and esterase specific activities of cell free extracts of different
strains of lactobacilli
Lb. _helveticus strain
Substrate
Lys
Pro
Met
Ala
Leu
Arg-Pro
Gly-Phe
Gly-Pro
O-Nitro-C4
P- Nitro-C4
O-Nitro-C6
P- Nitro-C6
0-Nitro C8
P- Nitro-C8
Lb. casei strain
G
H
I
J
K
L
M
u
A
B
c
D
E
T
8
0.2
7
8
8
3
0.5
2.2
0.05
0.12
0.02
0.08
0.04
0.04
8
0.1
3
7
6
2
0.2
0.1
0.03
0.2
0.01
0.08
0.01
0.06
9.3
0.3
9.4
19.1
10.3
4.6
0.7
3.5
0.1
0.2
0.04
0.6
0.01
0.07
8.6
0.26
3.2
7.2
5.2
3.6
0.28
1.3
0.06
0.1
0.03
0.1
0.007
0.001
3
0.3
1.3
2.5
2.6
1.5
0.1
1.8
0.1
0.8
0.04
1
0.01
0.001
10.8
0.26
4.1
8.2
7
2.6
0.5
1.4
0.09
0.2
0.02
0.5
0.002
0.02
8.4
0.4
8.7
11.8
10.5
4.2
0.48
2.8
0.1
0.3
0.06
0.9
0.007
0.02
2
0.02
1
0.6
2"
0.5
0.05
0.2
0.07
0.3
0.1
0.7
0.5
0.6
6
0.06
1
2
4
1
0.06
0.5
0.5
1.4
1.1
22
1.2
2.7
1
0.02
0.4
0.4
1.67
0.03
0.01
0.04
0.12
0.4
0.2
0.9
0.3
0.8
5
0.06
1
2
4
1
0.05
0.2
0.24
0.8
0.31
2.1
0.8
2.2
0.6
0.08
0.1
0.1
0.4
0.2
0.004
0.008
0.09
0.3
0.08
0.6
0.11
0.6
3.2
0.5
0.6
1.2
1.7
0.7
0.08
0.2
0.5
0.88
0.9
4.4
0.5
0.9
0.05
0.1
0.3
0.5
0.6
0.01
0.02
0.4
0.3
0.2
0.9
0.2
1.2
were generally higher for most Lb. helveticus strains
compared to Lb. casei strains. Three strains of Lb.
helveticus, G, I and M displayed the highest amino- and
dipeptidyl-aminopeptidase activity. Results in other re­
ports (13, 24, 31) demonstrated that most Lb. helveti­
cus strains exhibited greater aminopeptideise activities
than Lb. casei strains; In both species, aminopepti­
dases show greater specificity for Lys-pNA , Leu-pNA
and Ala-pNA substrates. The relative rate of hydrolysis
of dipeptidyl-aminopeptidase substrates varied from
strain to strain, but dipeptidyl-aminopeptidase generally
displayed higher specificity on Arg-Pro and Gly-Pro p­
nitroanalide than Gy-Phe. Strains I and M had the high­
est activity toward Arg-Pro followed by Gly-Pro and Gly­
Phe. Comparable results were reported by EL AB­
BOUD! eta/. (9) pointing to the presence of X-pro­
lyldipeptidyl-aminopeptidase based on the specific ac­
tivities of the substrates tested. A similar pattern on
these substrates was also found by GOMEZ eta/. (20),
but the enzyme activities detected in their study were
considerably lower than our results. Furthermore, the
results showed that Lb. helveticus strains exhibited 2­
15 fold higher dipeptidyl-aminopeptidase activity com­
pared to Lb. easeL These findings are consistent with
those described earlier on related microorganisms (13).
It was recently demonstrated that aminopeptidases
seem to play a role in the hydrolysis of bitter peptides (1,
2, 3) while dipeptidyl-aminopeptidase seems to be more
limited (B). Thus, the presence of Lactobacillus adjuncts
during cheese ripening may have a significant impact in
reducing bitterness.
Results in Table 1 show the esterolytic activity on o­
and p-nitrophenyl fatty acid derivatives. All Lactobacil­
lus strains tested in this study displayed intracellular
esterolytic activity, but the values were low for most of
the lactobacilli tested when compared to the peptidase
activitY. In contrast to the trend of peptidase activity, Lb.
casei strains displayed somewhat higher esterolytic activ­
ity than Lb. helveticus. These findings are contradictory
to the results of GOBBETTI eta/. (19). p-Nitrophenyl
derivatives appear to hydrolyze at a higher rate than o­
nitrophenyl derivatives with greater specificity toward p­
nitrophenyl caproate. EL-SODA eta/. (12) reported that
all Lactobacillus species hydrolyzed p-nitrophenyl esters
of short chain fatty acids to a measurable extent and as
general trend, the rate of hydrolysis increased with in­
crease in the number of carbon atoms in substrate.
KHALIO eta/. (22) also demonstrated that p-nitrophenyl
Milchwissenschaft 54 (2) 1999
1
R
2
0.05
0.5
0.5 1.2 0.7
0.01
0.4
0.12
0.3
0.1
1.7
0.09
0.3
derivatives are hydrolyzed at a significantly higher rate
when compared to o-nitrophenyl derivatives of fatty ac­
ids. The straight configuration of p-nitrophenyl deriva­
tives may make them more accessible to the enzyme's
active sites (22). Thus, the strains of lactobacilli evalu­
ated herein showed variable rate of active esterase sys­
tems that hydrolyzed o- and p-nitrophenyl butyrate, cap­
roate and caprylate. This suggests that esterases of
these strains could contribute at different extent to the
flavor development during cheese ripening process
through production of short chain fatty acids (12, 24, 31 ).
3.2 Autolytic properties of Lactobacillus strains
The results shown in Fig. 1 indicate considerable
variation in the rate of autolysis of different strains
tested. Lb. helveticus cultures could be divided in 2
groups. Highly autolytic strains (G, I, M, U and H) which
exhibited autolysis ranging from 55 to 80% and a sec­
ond group (L, K and J) which showed autolysis levels
ranging from 5 to 25% after 48 h of incubation. Lb. casei
strain C showed the highest level of autolysis (95% af­
ter 48 h of incubation), while strains T, D, B and R
formed an intermediate group with levels of autolysis
ranging from 40% to 65%. The lowest levels of autolysis
for Lb. casei were observed in strain E (20%) and A
(15%). Our results are comparable to previous work on
the autolytic properties of the lactobacilli (11, 26). In order
for an intracellular enzyme to play a meaningful role to
enhance cheese ripening, it has to be released in the
cheese matrix through autolysis. Thus, autolysis appears
to be an important criteria that should be considered in
selection of Lactobacillus species as adjuncts.
100.-------------------------------,
90
Lb. helveticus
80 if 70 "";;;" 60 -~50
£:::l 40
<(
30
20
10
0 ~;:::.o"'T--< F,.---.,::C)-,---,.----,-£¥;;::.=...,.----,----,--J
6 24 48
3
0
3
6 24 48 Time (h) 0
Fig.1:
Rate of autolysis of different strains of Lb. helveticus
(G, H, I, J, K, L, M, U) and Lb. casei (A, 8, C, D, E, R, T). •
Ag, H; e 8, K; A. C, J; + D, U; 0 E, I; 0 R, L; ~ T, M; G
*
EI-Soda, Cheese ripening
88
3.3 Release of intracellular enzymes during
autolysis of Lactobacillus cells
The rates of release of cellular enzyme activities
from different strains of Lactobacillus are shown in
Figs. 2 and 3. Some strains of lactobacilli showed in­
stant release of aminopeptidase activity which was de­
tected in a appreciable level at zero time after the cells
were suspended in phosphate buffer (Rg. 2). This was
then followed by a gradual increase of enzyme activity
release reaching its maximum after 24 h. High activity
release was detected in most of the Lactobacillus strains
after 3 h. Strain U showed the highest activity release ­
65% of the total activity after 3 h and 98% after 24 h.
Maximum values of enzyme activity released after 24 h
were 98, 86, 83 and 81% for strain U, M, C and G, re­
spectively. Most Lactobacillus strains showed a de­
crease of enzyme activity varying from 5 to 40% after
48 h incubation. This may indicate low stability of the
enzyme under the experimental conditions described. ·
100 90 ~ 80 5l<tt 70 ~ 60
(I)
;. 50 's;40 'E
30
<tt
a.. 20 < 10
0
~
~
I:.
c
f
,_,
;
I' I G M J
_.~ -
c
H L
u
K
Lb. helveticus strains
c
: l_i~
8 T D R A
Lb. casei strains
E
IOhD 3hD 6hD 24hE 48hllllllll
Fig. 2: Rate of release of aminopeptidase activity of different
strains of lactobacilli after different times of incuba­
tion at 30°C in buffer system. Lysine p-nitroanilide
was used as substrate
~ 80 -; 70 "'as (jj
6o
;. 5 0
<tt 30
~
20
0
~ 10 I~ ~ ~ Jl ~
O
I
G M J
Jil ,J
H L U K
Lb. helveticus strains
I
0 hD
3hD
6 hD
4. Conclusion
It is possible to differentiate the adjunct cultures
based on the following criteria: total peptidase and total
esterase activities,·% autolysis and rate of peptidase
and esterase release. Among the Lb. he/veticus ad­
juncts, strain I is the only strain showing high levels of
peptidase and esterase activities, high autolysis, and
high enzyme release. Although strain U is comparable
to I in autolysis and rate of peptidase release, it exhib­
ited very little esterase and peptidase activity.
In cultures R and D low levels of both peptidase and
. esterase activity were measured in the crude cell free
extract. They were resistant to autolysis and showed
little enzyme release. These cultures are not expected
to have a significant impact during cheese ripening.
Despite the fact that Lb. helveticus Land Lb. caseiE
produce relatively high levels of peptidase and esterase
activity, their role during cheese ripening is also ex­
pected to be minimal because very little enzyme activity
will be released in the cheese matrix due to their high
resistance to cell lysis.
This study shows that it is valuable to screen adjunct
cultures for their autolytic properties in addition to their
proteolytic and lipolytic activities. These characteristics
could be useful in predicting efficacy of selected com­
mercial adjunct cultures to enhance cheese quality.
Acknowledgements
Research financial support by Dairy Management, Inc.
and California Research Foundation is gratefully acknow­
ledged.
~40
0
0
decrease of activity was noticed after 48 h of incuba­
tion, which may be related to a higher stability of the
esterase system of these microorganisms. Contrary to
peptidase, the release of esterases from Lb. casei strains
was higher than from Lb. helveticus strains. The results
revealed that the rates of esterase release were con­
siderably lower than rates of peptidase activity release.
Lb. helveticus I and Lb. casei C showed high levels of
enzyme release and demonstrated high levels of auto­
lysis. On the other hand, Lb. helveticus A and Lb. casei E,
which were resistant to the autolytic process, released very
little enzymatic activities. The results reveal the impor­
tance of autolysis in the release of cellular enzymes. This
process is expected to be of major importance during
cheese ripening since the released enzymes will actually
participate in the breakdown of fats and proteins which
contribute to cheese flavor development (5, 6, 7, 11, 26).
m
jll
~ ~
,J
.d
CBTDRAE Lb. casei strains 24 h - 48 h IIIII
I
Fig. 3: Rate of release of esterolytic activity of different strains
of lactobacilli after different times of incubation at
3oac in buffer system. p-Nitrophenyl caproate was
used as substrate
·
Esterase release from the different Lactobacillus
species tested followed a different pattern (Fig. 3) com­
pared to peptidases. No instant release of esterase ac­
tivity could be measured in any of the strains tested. No
4. References
(1) ARDO, T., LARSSON, P., MANSSON, L., HEDEN­
BERG, A: Milchwissenschaft 44 485-490 (1 ~89)
(2) BAANKRIES, R.: PhD Thes. Univ. Amsterdam (1992)
(3) BARTELS, H., JOHNSON, M., OLSON, N.: Milch­
wissenschaft 42 139-144 (1987)
(4) BRANDL, E., ZIZER, T.: Osterr. Milchw., Wiss. Beil.
2815-24(1973)
.
(5) CHAPOT-CHARTIER, M.P.,· DENIEL, C., ROUS­
SEAU, M., VASSAL, L, GRIPON, J.C.: Int.Dairy J. 4
251-269(1994)
'
(6) CHAPOT-CHARTIER, M.P.: Lait7691-109(1996).
(7) CROW, V.L, COOLBEAR, T., GOPAL, P.K., MART.;
LEY, F.G., McKAY, L.L., RIEPE, H.: Int. Dairy J. S
. 855-875 (1995)
'
Milchwissenschaft 54 (2) 1999
EI-Soda, Cheese ripening
89
(8) El ABBOUD!, M., EL SODA, M., JOHNSON, M., OL­
SON, N.F., SIMARD, R.F., PANDIAN, S.: Milchwis­
senschaft 47 625-628 (1992)
(9) El ABBOUD!, M., EL SODA; M., PANDIAN, S.,
BARREAU, M., TREPANIER, G., SIMARD, R.E: Int.
DairyJ. 255-64(1991)
(10) EL SODA, M., DESMAZEAUD, M.J.: Can. J. Micro­
bial. 281181-1188 (1982)
(11) EL SODA, M., FARKYE, N., VUILLEMARD, J.C.,
SIMARD, R.E., OLSON, N.F., EL KHOLY, W., DAKO,
E., MEDRANO, E., GABER, M., LIM, L: In Food fla­
vor: generation, analysis and process influences (Ed.
G. Charalarnbous). 2205-223 Elsevier Sci. (1995)
(12) EL SODA, M., LAW, J., TSAKALIDOU, E., KA­
LANTZOPOULOS, G.: In Food flavor: generation,
analysis and process influences (Ed. G. Charalam­
bous) 1823-1184, Elsevier Sci. (1995)
{13) EL SODA, M., MACEDO, A., OLSON, N.: Milchwis­
senschaft 46 358-366 (1991)
(14) EL SODA, M.: In Microbiology and Biochemistry of
cheese and fermented milk (Ed. B. Law) 219-246,
BlackieAcad. & Prof. (1993)
(15) FOX, P.F., LAW,J.: FoodBiotechnol.523~262(1991)
(16) FOX, P.F., lAW, J., McSWEENEY, P.LH., WALLACE,
J.: In Cheese: Chemistry, Physics and Microbiology,
Vol.1 (Ed.P.F. FOX)439-470, Chapman&Hall (1993)
(17) FOX, P.F., WALLACE, J.M., MORGAN, S., LYNCH,
C.M., NILAND, E.J., TOBIN, J.: Ant. v. Leuwenhoek
70271-297(1996)
(18) FREY, J.P., MARTH, E.H., JOHNSON, M.E., OL­
. SON, N.F.: Milchwissenschaft41681-685 (1986)
(19) GOBBETTI, M., FOX, P.F., STEPANI!\K, L.: ltal. J.
Food Sci.2127-135 (1996)
(20) GOMEZ, M.J., GAYA, P., NUNEZ; M., MEDINA, M:
Milchwissenschaft 51 315-319 (1995)
(21) JOHNSON, J., ETZEL, M., CHEN, C., JOHNSON,
M.: J. Dairy Sci. 78 769-776 (1995)
(22) KHALID, N.M., EL SODA, M., MARTH, E. H.: J. Dairy
Sci. 732711-2719(1990)
(23) KHALID, N.M., EL SODA, M., MARTH, E. H.: J. Dairy
Sci. 7429-45 (1991)
(24) KHALID, N.M., MARTH, E.H.: J. Dairy Sci. 73 2669­
2684(1990)
.
(25) LANE, C.N., FOX, P.F.: Int. Dairy J. 6 715-728 (1996)
133­
(26) LORTAL, S., LEMEE, R., VALENCE, F.: Lait
150(1997)
(27) LOWRY, 0., ROSEBROUGH, A., FARR, L., RAN­
DALL, R.: J. Bioi. Chern. 193 265-275 (1951)
'(28) MARTLEY, F.G., CROW, V.L.: Int. Dairy J. 3 461­
484(1993)
(29) McSWEENEY, P.L.H., FOX, P.F., LUCEY, K.N.,
JORDAN, K.N., COAGAN, T.M.: Int. Dairy J. 3 613­
634(1993)
(30) OHMIYA, K., SATO, Y.: Agric: Bioi. Chern. 39 585­
589(1975)
(31) PETERSON, S.D., MARSHALL, R.T.: J. Dairy Sci.
731395-1410 (1990)
'
(32) THIBOUTOT, H., DAKO, E., EL SODA, M., VUIL­
LEMARD, J.C., POWER, N., SIMARD, R.E.: Milch­
wissemschaft 50 448-452 ( 1995)
(33) WILKINSON, M.G.: In Cheese: Chemistry, Physics
and Microbiology, Vol. 1 (Ed. P.F. FOX) 523-556,
Chapman & Hall ( 1993)
n
Milchwissenschaft 54 (2) 1999
5. Summary
EL-SODA, M., MADKOR, S.M., TONG, P.S.: Evalua­
tion of commercial adjuncts for use in cheese ripen­
ing: 1. Enzymatic activities and autolytic properties of
freeze-shocked adjuncts in buffer system. Milchwis­
senschaft 54 (2) 85-89 (1999).
51 Cheese ripening (commercial adjuncts)
Eight strains of Lb. helveticus and 7 strains of Lb. casei
adjunct cultures were compared according to intracellular
aminopeptidase and dipeptidyl-aminopeptidase activities
using synthetic p-nitroanilides derivatives of amino acids
and dipeptides. Esterolytic activity was measured by using
p- and o-nitrophenyl derivatives of fatty acids. The auto­
lytic properties of the cells and their intracellular peptidase
and esterase release were also evaluated.
Lactobacilli showed different aminopeptidase, dipepti­
dyl-arninopeptidase and esterolytic activities which were
species- and strain-specific. Among the adjuncts tested,
Lb. helveticus I, G, and M, and Lb. caseiC had high enzy­
matic potentials. Their specific activities for lysine amino­
peptidase varied from 5 to 10; while the level of esterase
activity on p-nitrophenyl caproate ranged from 0.6 to 2.0.
The release of these enzymes during autolysis in buffer
system ranged from 60 to 80% after incubation at 30
for
48 h. Lb. casei A, E and Lb. helveticusJ exhibited high en­
zyme activity but their rate of autolysis was low (2 to
20%).This study demonstrates a means to classify adjunct
cultures according to their levels of autolysis, enzyme ac­
tivity, and rate of enzyme release.
oc
EL-SODA, M., MADKOR, S.M., TONG, P.S.: Sewer­
tung handelsi.iblicher Zusatze fi.ir die Verwendung bei
der Kasereifung: 1. Enzymatische Aktivitat und autoly­
tische Eigenschaften von gefriergeschockten Zusat­
zen im Puffersystem. Milchwissenschaft 54 (2) 85-89
(1999).
51 Kasereifung (Zusatze)
8 Stamme von Lactobacillus helveticus- und 7 von
Lb.casei-Zusatzkulturen wurden auf intrazellulare Amino­
peptidase- und Dipeptidyi-Aminopeptidase-Aktivitat unter
Verwendung synthetischer p-Nitroanilidderivate von Ami­
nosauren und Dipeptiden verglichen. Die esterolytische
Aktivitat wurde mithilfe von p- und o-Nitrophenylderivaten
von Fettsauren gemessen. Auch die autolytischen Eigen­
schaften der Zellen und ihre intrazellulare Peptidase- und
Esterasefreisetzung wurden bewertet.
Laktobazillen zeigten unterschiedliche Aminopeptidase-,
Dipeptiyi-Aminopeptidase- und esterolytische Aktivitaten,
die species- und stammspezifisch waren. Bei den gepruf­
ten Zusatzen hatten Lb. helveticus I, G,und M und Lb.
casei C hohe enzymatische Potentiale. lhre spezifischen
Aktivitaten bei Lysinaminopeptidase lagan zwischen 5 und
10, wah rend der Level der Esteraseaktivitat bei p-Nitro­
phenylcaproat zwischen 0,6 und 2,0 lag. Die Freigabe die­
ser Enzyme wah rend der Autolyse im Puffersystem betrug
60-80% nach lnkubation bei 30°C fur 48 h. Lb. casei A,E
und Lb. helveticus J wiesen eine hohe Enzymaktivitat auf,
jedoch war ihr Autolysegractniedrig (2-20%). Diese Studie
zeigt eine Moglichkeit auf, Zusatzkulturen nach Auto lyse­
level, Enzymaktivitat und Grad der Enzymfreisetzung ein­
zuordnen.