Title: Use of strains of Streptococcus thermophilus which are incapable of hydrolyzing urea in dairy products
Abstract: The invention relates to the use of at least one strain of Streptococcus thermophilus which is incapable of hydrolyzing urea in the manufacture of cheese or fermented dairy products such as yogurts in order to obtain an acidification kinetic which is independent from the content of various components of the milk.
Patent Number: 6,962,721 Issued on 11/08/2005 to Sepulchre, et al.
| Inventors:
|
Sepulchre; Anne-Marie (Saint-Avertin, FR);
Monnet; Christophe (Plaisir, FR);
Corrieu; Georges (Viroflay, FR)
|
| Assignee:
|
Texel (St. Romain, FR);
Institut National de la Recherche Agronomique (Paris, FR)
|
| Appl. No.:
|
088350 |
| Filed:
|
September 15, 2000 |
| PCT Filed:
|
September 15, 2000
|
| PCT NO:
|
PCT/FR00/02577
|
| 371 Date:
|
June 14, 2002
|
| 102(e) Date:
|
June 14, 2002
|
| PCT PUB.NO.:
|
WO01/22828 |
| PCT PUB. Date:
|
April 5, 2001 |
Foreign Application Priority Data
| Current U.S. Class: |
426/43; 426/61; 426/582; 426/583 |
| Intern'l Class: |
A23C 009/12 |
| Field of Search: |
426/36,42,43,61,582,583
435/885,253.4
|
References Cited [Referenced By]
U.S. Patent Documents
| 5116737 | May., 1992 | McCoy.
| |
| 6056979 | May., 2000 | Benbadis et al.
| |
| Foreign Patent Documents |
| WO 96/1062/7 | Apr., 1996 | WO.
| |
Other References
W. Tinson, "Metabolism of streptococcus thermophilus," The Australian Journal
of Dairy Technology, vol. 37, No. 1, 1982, pp. 17-21.
B. Bianchi Salvadori, "Characteristics of some streptococcus thermophilus strains
for the preparation of starters dehydrated . . . ", Scienza E. Tecnica Lattiero-Cassearia,
vol. 34, No. 4, 1983, pp. 227-248.
A. Zourari, "Caracterisation de bacteries lactiques thermophiles isoleos de yaourts
artisanaux grecs," Le Lait, vol. 77, No. 4, 1991, pp. 445-461 (Not translated).
V. Juillard, "Mise en evidence d'uno activite ureasique chez Streptococcus thermpohilus,"
Canadian Journal of Microbiology, vol. 34, No. 6, 1998, pp. 818-822.
Stoyanov et al., Neuchni Trudove, Vissh Institut po Khraniteine i Vkusona
Promishlenost (1995), 40:211-217, Plovid, Bularia, with full English translation.
Other Document: May 25, 2004 e-mail from Henk Spierenburg regarding S. thermophilus
CNRZ 407 strain.
|
Primary Examiner: Hendricks; Keith
Attorney, Agent or Firm: Burns, Doane, Swecker & Mathis, LLP
Claims
1. A method for obtaining, during the manufacture of a dairy product selected
from the group consisting of cheeses and other fermented dairy products, an acidification
kinetic which is substantially independent of the content of the milk in terms
of constituents which are involved in the metabolism of urea, said method comprising
incorporating with the milk at least one strain of
Streptococcus thermophilus
which is incapable of hydrolyzing urea.
2. The method according to claim 1, in which the acidification kinetic is substantially
independent of the urea content of the milk.
3. The method according to claim 1, in which the acidification kinetic of the
milk is substantially independent of the nickel or cobalt content of the milk.
4. The method according to claim 1, in which the acidification kinetic of the
milk does not exhibit any temporary slowing down.
5. A method according to claim 1, in which there is incorporated with the milk
at least one mutant strain of
Streptococcus thermophilus which is incapable
of hydrolyzing urea, at a seeding rate lower than the seeding rate used for the
parent strain of
Streptococcus thermophilus capable of hydrolyzing urea.
6. A method according to claim 1, in which the
Streptococcus thermophilus
strain is the strain 298-K registered at the CNCM under number I-2311.
7. The method according to claim 1, in which the
Streptococcus thermophilus
strain is the strain 298-10 registered at the CNCM under the number I-2312.
Description
The present invention relates to controlling the acidification kinetic of milk
during the manufacture of cheeses or fermented milks such as yoghurts, through
the use of
Streptococcus thermophilus bacteria which are at least partially,
preferably totally, incapable of hydrolyzing urea.
Streptococcus thermophilus is a thermophilic lactic bacterium used as
a lactic ferment in the dairy industry. Used first of all for the manufacture of
fermented milks such as yoghurt, it is now increasingly used in cheese production.
This bacterium converts lactose into lactic acid, and through this has an acidifying
activity. In the case of cheeses notably, this acidification not only encourages
the action of the rennet and the synaeresis of the curds but also inhibits the
growth of many undesirable bacteria, certain of which are pathogenic bacteria,
and even allows their elimination at a greater or lesser speed.
The acidifying activity of this bacterium is however accompanied by a urea hydrolysis
activity, an activity that affects the acidification kinetic. Tinson et al (1982a)
showed that the urea hydrolysis reaction, giving-carbon dioxide and ammonia, resulted
in a temporary decrease in the acidification speed, measured by means of a pH probe.
The authors of this article conclude therefrom that the changes in pH cannot be
used to measure the lactic acid production in
S. thermophilus cultures,
since the results that would be obtained would be erroneous owing to the production
of ammonia. Furthermore, Spinnier and Corrieu in 1989 observed that the addition
of urea led to a drop in the acidification speed.
On an industrial scale, the hydrolysis of urea by
Streptococcus thermophilus
poses a number of problems.
This is because, in cheese manufacturing for example, the technological operations
(cutting of the curds, stirring, etc.) must take place at given values of pH, but
in practice these operations are generally carried out at predetermined times.
Therefore the variations in acidifying activity due to urea hydrolysis lead to
defects and significant variability in the cheeses (texture, moisture level, ripening).
Martin et al (1997) thus observed that the variations in urea content caused changes
in the acidification kinetics and in the texture of Reblochon type cheeses, confirming
the results obtained by Spinnier and Corrieu (1989).
Moreover, the production of ammonia increases the time necessary to reach
a given pH. This results in the equipment being tied up for longer and in an increase
in the risk of contamination by undesirable micro-organisms.
Furthermore, it is desirable that the cheese-making whey does not contain
an excessive amount of ammonia, since this whey is often used in animal feed.
This phenomenon is difficult to control, notably since the urea content of milk
is variable (generally from 2 to 8 mM) and depends in particular on the feeding
of the livestock. To overcome this problem, Martin et al (1997) proposed measuring
the urea content of the milk and then adapting the manufacturing parameters. However,
the use of such a urea quantitative analysis system would be highly constraining,
and would not in any case resolve the drawbacks due to slowing down of the acidification
speed in the presence of urea (equipment tied up for a longer time, increase in
the risks of contamination, etc.) and to a high ammonia content of the whey.
The authors of the present invention have revealed that the use of
Streptococcus
thermophilus strains not, or not totally, hydrolyzing urea, as lactic ferments
in the production of dairy products, made it possible to solve the aforementioned
problems. These strains are designated "ur(-) strains" in the remainder of this application.
Until now, the only ur(-)
Streptococcus thermophilus strains described
are the CNRZ 407 strain (Juilliard et al, 1988) and the mutant strain isolated
by Tinson et al (1982b). However, the information known relating to these two strains
does not allow the technological importance of ur(-) strains to be realized.
One object of the present invention is therefore the use of at least one strain
of
Streptococcus thermophilus which is at least partially, preferably totally,
incapable of hydrolyzing urea, during the manufacture of cheeses or fermented dairy
products such as yoghurts, in order to obtain an acidification kinetic which is
substantially independent of the content of the milk in terms of its constituents.
Within the context of the present invention, "the acidification kinetic" means
the variation in pH of the fermentation medium as a function of time.
"Content of the milk in terms of its constituents" means in particular the
urea content of the milk, which differs from one milk to another, depending on
the origin of the animal or its feed. It also means the content of the milk in
terms of other constituents which are involved in the metabolism of urea. Amongst
these constituents can be cited for example nickel or cobalt. These constituents
may be present naturally in the raw material used (the milk) or may have been added.
Another object or the invention is a method for obtaining, during the manufacture
of cheeses or fermented dairy products such as yoghurts, an acidification kinetic
which is substantially independent of the content of the milk in terms of its constituents,
in which there is incorporated with the milk at least one strain of
Streptococcus
thermophilus which is at least partially, preferably totally, incapable of
hydrolyzing urea.
The ur (-)
Streptococcus thermophilus strains used in accordance with
the present invention can be obtained by a mutagenic treatment or by spontaneous
mutation, or also be isolated in nature.
The strains 298-K and 298-10, which are respectively a spontaneous mutant and
a mutant obtained after mutagenic treatment, were registered at the CNCM on 14
Sep. 1999 under the numbers I-2311 and I-2312, respectively.
Any ur(-) strain cultured according to the protocol of Tinson et al (1982b),
or preferably according to the protocol described in Example I, can also be used.
The ur (-)
Streptococcus thermophilus strains can be used alone or in
a mixture with other micro-organisms such as
lactococci, lactobacilli, or
any other micro-organism usable in the dairy industry.
The authors of the present invention have shown that the importance of the ur(-)
Streptococcus thermophilus strains is multifaceted. In fact, they have revealed
that the ur(-) mutants make it possible not only to have control over the variations
in acidification kinetics, but that they are moreover stable and exhibit good growth
in milk.
Furthermore, the ur(-) strains make it possible to obtain acidification
kinetics of milk which are regular, do not exhibit any temporary slowing down,
and are a function of the area concentration, unlike the kinetics observed with
the ur(+) strains.
The ur(-) strains do not produce ammonia during their growth in milk, which is
advantageous from the point of view of using the whey in animal feed.
Finally, the strains selected for their ur(-) phenotype surprisingly have
variable acidifying characteristics, compared with the acidification kinetics observed
with the parent strains.
"Variable acidification kinetic" means an acidification kinetic which is
for example faster or slower compared with the acidification kinetics observed
with the parent strains. "Heterogeneity" between the acidification kinetics of
the different ur(-) mutants with regard to the parent strains can also be spoken of.
Another object of the invention is therefore a method of selecting
Streptococcus
thermophilus strains useful during the manufacture of cheeses or fermented
dairy products, in which mutant strains of
Streptococcus thermophilus which
are at least partially, preferably totally, incapable of hydrolyzing urea, allowing
an acidification kinetic to be obtained which is substantially independent of the
content of the milk in terms of its constituents, are selected for their ability
to acidify a milk according to acidification kinetics which are variable compared
with the acidification kinetics of the parent strains.
In general terms, the choice of the acidifying properties of the ur(-) strains
can be made as a function of the cheese or fermented milk manufacturing technology
for which these strains are used.
Thus, certain ur(-) strains are characterised more particularly by an absence
of the post-acidification phenomenon.
For other strains, the time necessary to reach a given pH proves to be shorter
than for the parent ur(+) strains. Thus, this property makes it possible to seed
the milk with a ur(-) mutant strain at a rate lower than the rate generally used
for the parent ur(+) strain. This rate can be around 25%, perhaps even around 50%
lower compared with the rate that would be used for the parent strain.
One object of the present invention is therefore a method according to the invention,
in which there is incorporated with the milk at least one mutant strain of
Streptococcus
thermophilus which is at least partially, preferably totally, incapable of
hydrolyzing urea, at a seeding rate lower than the seeding rate used for the parent
strain of
Streptococcus thermophilus capable of hydrolyzing urea.
The figures and examples below illustrate the invention without limiting the
scope thereof.
LEGEND FOR THE FIGURES
FIGS. 1A-1B depicts acidification curves for reconstituted skimmed milk, obtained
with the ur(+) strain RD298 and with the spontaneous ur(-) mutants (FIG. 1A) or
those obtained after treatment with NTG (FIG. 1B).
FIGS. 2A-2B depicts the acidification curves for reconstituted skimmed milk,
obtained with the strain ST888 and with the spontaneous ur(-) mutants (FIG. 2A)
or those obtained after treatment with NTG (FIG. 2B).
FIGS. 3A-3B depicts the acidification curves for UHT skimmed milk, obtained
with the strain RD298 and with the spontaneous ur(-) mutants (FIG. 3A) or those
obtained after treatment with NTG (FIG. 3B)
FIGS. 4A-4B depicts acidification curves for UHT skimmed milk, obtained with
the strain ST888 and with the spontaneous ur(-) mutants (FIG. 4A) or those obtained
after treatment with NTG (FIG. 4B).
FIGS. 5A-5C depicts the acidification curves obtained with the strain RD298
(FIG. 5A) and the ur(-) mutants RD298-K (FIG. 5B) and RD298-10 (FIG. 5C), on UHT
skimmed milk supplemented with different amounts of urea.
FIGS. 6A-6C depicts the acidification curves obtained with the strain RD298
(FIG. 6A) and the ur(-) mutants RD298-K (FIG. 6B) and RD298-10 (FIG. 6C), on UHT
skimmed milk supplemented or not with nickel (10 μg/l of NiSO
4.7H
2O).
FIG. 7 depicts the acidification curves obtained with the strain RD672 and ur(-)
mutants originating from this strain, on reconstituted skimmed milk.
EXAMPLES
Example 1
Method of Culturing Ur(-) Bacteria on Petri Dishes.
An agar-based medium whose composition is shown in Table 1 is prepared and poured
into Petri dishes of diameter equal to 9 cm.
| TABLE 1 |
| Composition of the culture medium. |
| |
| |
Tryptonea |
2.5 |
g |
| |
Pepsic meat peptonea |
2.5 |
g |
| |
Papainic soya peptonea |
5 |
g |
| |
Autolytic yeast extractb |
2.5 |
g |
| |
Meat extracta |
5 |
g |
| |
Sugar (glucose, lactose or saccharose) |
5 |
g |
| |
Sodium glycerophosphate.6H2O |
19 |
g |
| |
Magnesium sulphate |
0.25 |
g |
| |
Ascorbic acid |
0.5 |
g |
| |
Agar |
15 |
g |
| |
Distilled, water |
1 |
litre |
| |
| |
aBlokar company |
| |
bFischer Scientific company |
If need be, a cofactor of urease can be added to this medium. Adjust the pH to
7.0 and autoclave for 15 minutes at 115° C.
The
St. thermophilus cells to be analyzed are seeded on this medium so
as to obtain around 100 colonies per Petri dish. The cultures take place under
anaerobic conditions at a temperature of 35-45° C., preferably 37-42° C.
After two days of culture, there is poured over each Petri dish around 20 ml
of an agar-based solution prepared as follows: dissolve by heating 15 g of agar
in 1 liter of a potassium phosphate buffer solution at 50 mM (pH 6) supplemented
with 100 mg/l of bromothymol blue, cool the solution to 50° C., add 10 g of
urea and acidify the medium with hydrochloric acid until a yellowish-orange colour
is obtained.
After solidification of the agar, the Petri dishes are incubated for 1 hour
at 37° C. The ur(+) clones form blue-coloured halos owing to the production
of ammonia, whereas the ur (-) clones form yellow colonies. When the ur(-) mutants
are sought, the clones not forming a blue halo are recovered and tested again on
the same culture medium in order to confirm the ur(-) characteristic. It should
also be verified that these mutants do not consume urea (or consume it only partially)
when they are cultured in milk.
Example 2
Selection of Mutants for the Metabolism of Urea.
Mutants not consuming urea, or consuming it slightly, were sought from the
RD298, RD672 and STS888 strains of
St. thermophilus. Two approaches were
used. In the first approach, the mutants were sought after treatment with a mutagenic
agent, while in the second approach, spontaneous mutants were sought.
a) Selection by Means of a Mutagenic Agent
The mutagenic treatment is carried out as described below.
The strains are cultured at 42° C. in 5 ml of M17 culture medium (Terzaghi
and Sandine, 1975). The culture is stopped at the end of the exponential phase,
and the cells are recovered by centrifuging and then washed with 100 mM (pH 7)
phosphate buffer. The cells are next recovered in 1 ml of buffer containing a variable
content of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and incubated for
1 hour at 42° C. The cells are next washed twice with 5 ml of buffer and seeded
on the culture medium so as to obtain around 100 colonies per Petri dish. The culture
is carried out as described previously (Example 1). Table 2 describes the results
obtained during 3 mutageneses.
| TABLE 2 |
| Selection of ur(-) mutants after treatment with a |
| mutagenic agent (NTG). |
| |
|
Viability |
|
|
|
| |
|
(% of |
| |
NTG |
cells |
Number |
Number |
| St. |
concen- |
having |
of |
of ur(-) |
Proportion |
| thermophilus |
tration |
survived |
colonies |
clone |
of ur(-) |
| strain |
used (μg/ml) |
the NTG) |
cultured |
obtained |
clones (%) |
| ST888 |
20 |
10 |
980 |
11 |
1.1 |
| ST888 |
5 |
48 |
1000 |
5 |
0.5 |
| RD672 |
50 |
41 |
10600 |
41 |
0.4 |
| RD298 |
50 |
16 |
3200 |
15 |
0.5 |
b) Selection of Spontaneous Mutants
In a population of micro-organisms, there often exist spontaneous mutants for
a gene or a given characteristic. This type of mutant is of great interest, since
the fact that no mutagenic agent has been used eliminates the risk of causing non-sought-after
mutations (mutations other than for the characteristic studied), which might impair
the technological abilities of the strains. However, the frequency of spontaneous
mutants within a population for a given characteristic is generally very low, of
the order of 1 in 1 million (variable depending on the strain and characteristic).
Therefore the selection of spontaneous mutants generally requires either the development
of a method making it possible to culture a very high number of clones, or the
definition of a procedure for enriching mutants. No procedure for enriching ur(-)
mutants has a priori been described. Moreover, given that the procedure of culturing
on Petri dishes does not allow the analysis of more than 100 colonies of
St.
thermophilus per dish, the selection of spontaneous mutants might have been
expected to be unfeasible, since it would have been necessary to culture several
thousand, perhaps even tens of thousands, of Petri dishes, in order to have chances
of isolating a spontaneous mutant. However, the authors of the present invention
noticed that, in the
St. thermophilus cultures, the proportion of spontaneous
ur(-) mutants was high (around 1 in 2500 for ST888, 1 in 4000 for RD672 and 1 in
1200 for RD298), and that it is therefore possible to easily isolate this type
of mutant (Table 3).
| TABLE 3 |
| Selection of spontaneous ur(-) mutants. The protocol |
| used is the same as that described in paragraph a) "selection |
| by means of a mutagenic agent", except that the mutagenic |
| agent is omitted. |
| |
St. |
Number of |
Number of |
Proportion of |
| |
thermophilus |
colonies |
ur(-) clones |
ur(-) clones |
| |
strain |
cultured |
obtained |
(%) |
| |
| |
ST888 |
16000 |
6 |
0.04 |
| |
RD298 |
7400 |
6 |
0.08 |
| |
RD672 |
24000 |
6 |
0.03 |
| |
47 of the 90 mutants obtained were studied. The results relating to stability,
enzymatic characterization and the acidifying behaviour of these mutants are described below.
Example 3
Properties of the Ur(-) Mutants.
a) Stability of the Mutants
In order to be able to be usable in an industrial context, the ur(-) mutants
must
be stable. However, no data existed as regards the stability of ur(-) mutants of
St. thermophilus. The authors of the present invention studied the stability
of 47 mutants originating from the strains ST888, RD672 and RD298. The strains
were subcultured daily in 10 ml of M17 culture medium, for 20 days. The cultures
were inoculated at 1% and incubated at 42° C. The set of 20 subcultures represents
around 130 generations. After the 20
th subculture, the strains were
seeded in milk and it was determined whether or not they consumed urea (cultures
of 15 hours at 42° C.). The results are shown in Table 4. It should be noted
that the ur(-) mutants, whether they are obtained by a mutagenic treatment or are
spontaneous mutants, are highly stable. In fact, only two reversions were detected
for the 47 mutants tested.
| TABLE 4 |
| Study of the stability of the ur(-) mutants. The |
| urea consumption was tested during cultures on milk, after 20 |
| successive subcultures in M17 culture medium. |
| St. |
|
Number of |
Number of mutants |
| thermophilus |
|
ur(-) mutants |
consuming urea after |
| strain |
Mutation |
tested |
20 subcultures |
| ST888 |
NTG |
6 |
1 |
| ST888 |
Spontaneous |
6 |
0 |
| RD298 |
NTG |
5 |
0 |
| RD298 |
Spontaneous |
6 |
0 |
| RD672 |
NTG |
19 |
0 |
| RD672 |
Spontaneous |
5 |
1 |
| Total |
/ |
47 |
2 |
b) Enzymatic Characterisation of the Mutants
The strains studied were cultured for 24 hours, under anaerobic conditions and
at 37° C., in a liquid culture medium whose composition is shown in Table
5. The cells were recovered by centrifuging, washed in buffer (HEPES 50 mM-EDTA
1 mM, pH 7.5), and then recovered in a volume of buffer representing 2% of the
volume of the culture. The ureasic activity was then measured on acellular extracts
(treatment of the cells in a ball mill and recovery of the supernatant from centrifuging
for 5 minutes at 20,000 g).
| TABLE 5 |
| Composition of the culture medium used for preparing |
| the extracts. |
| |
| |
Tryptonea |
10 |
g |
| |
Autolytic yeast extractb |
5 |
g |
| |
Sodium glycerophosphate.6H2O |
19 |
g |
| |
Ascorbic acid |
500 |
mg |
| |
Magnesium sulphate |
250 |
mg |
| |
Nickel sulphate.7H2O |
10 |
mg |
| |
Glucose |
10 |
g |
| |
Distilled water |
1 |
litre |
| |
| |
aBlokar company |
| |
bFischer Scientific company |
Adjust the pH to 7.0 and autoclave for 15 minutes at 115° C.
The ureasic activity measurements were carried out at 37° C., in HEPES 50
mM—EDTA 1 mM (pH 7.5) buffer. The reaction is triggered by the addition of
25 mM of urea, and the ammonia produced in 20 minutes is analyzed quantitatively,
using Nessler's reagent. The results are expressed in units (U) of urease activity
(one unit corresponds to one micromole of ammonia produced per minute) per milligram
of protein.
Table 6 shows the activity values obtained. The ur(-) mutants did not exhibit
any detectable ureasic activity, with the exception of the mutants 298-3.17 and
888-1.5. These correspond to mutants having a ur(+) phenotype in the presence of
nickel and a ur(-) phenotype in the absence of this compound. Now, the culture
medium used for preparing the acellular extracts contained nickel sulphate. In
these two strains, the mutation probably focuses on the nickel transport system
or the system allowing its incorporation into the active site of the urease.
These strains of
St. thermophilus could also exhibit a ur(-) phenotype
on account of an inability to transport urea. Such strains would therefore always
possess a measurable ureasic activity in acellular extracts.
| TABLE 6 |
| Measurement of the ureasic activity of acellular |
| extracts obtained from the parent strains and from the ur(-) |
| mutants. |
| Parent |
Ureasic |
Parent |
Ureasic |
Parent |
Ureasic |
| strain |
activity |
strain |
activity |
strain |
activity |
| Mutant |
(U/mg) |
Mutant |
(U/mg) |
Mutant |
(U/mg) |
| RD298 |
0.94 |
RD672 |
1.08 |
ST888 |
0.95 |
| 298-10 |
N.D. |
672-18(0) |
N.D. |
888-A |
N.D. |
| 298-K |
N.D. |
672-47(0) |
N.D. |
888-B |
N.D. |
| 298-I |
N.D. |
672-54(0) |
N.D. |
888-C |
N.D. |
| 298-J |
N.D. |
672-19(0) |
N.D. |
888-D |
N.D. |
| 298-L |
N.D. |
672-31(0) |
N.D. |
888-1 |
N.D. |
| 298-M |
N.D. |
672-59(50) |
N.D. |
888-2 |
N.D. |
| 298-N |
N.D. |
672-62(50) |
N.D. |
888-2.6 |
N.D. |
| 298-3.9 |
N.D. |
672-61(50) |
N.D. |
888-2.11 |
N.D. |
| 298-3.3 |
N.D. |
672-33(50) |
N.D. |
888-2.9 |
N.D. |
| 298-3.16 |
N.D. |
672-55(50) |
N.D. |
888-1.13 |
N.D. |
| 298-3.17 |
0.58 |
672-53(50) |
N.D. |
888-1.8 |
N.D. |
| |
|
672-70(50) |
N.D. |
888-1.5 |
0.42 |
| |
|
672-20(50) |
N.D. |
| |
|
672-50(50) |
N.D. |
| |
|
672-34(50) |
N.D. |
| |
|
672-22(50) |
N.D. |
| |
|
672-24(50) |
N.D. |
| |
|
672-10(50) |
N.D. |
| |
|
672-36(50) |
N.D. |
| |
|
672-60(50) |
N.D. |
| |
|
672-21(50) |
N.D. |
c) Acidifying Behaviour of the Mutants
In order to demonstrate the technological importance of the ur(-) strains, the
authors of the invention compared their acidifying characteristics with those of
the corresponding parent strains.
The following results were observed:
- unlike the parent strains, the ur(-) mutants do not exhibit a temporary
slowing down of the acidification speed due to hydrolysis of the urea; their acidification
curves are therefore more regular;
- the kinetics of acidification of the milk by the ur(-) mutants are little
affected or not affected by the urea, nickel and cobalt contents;
- furthermore, a high variability of the acidifying activities between
the ur(-) mutants is observed, compared with the acidifying activities of the parent strains.
A breakdown of the results obtained is shown below. The cultures were seeded
at
1% with a preculture carried out on sterilized reconstituted skimmed milk, then
cultured at 37° C.
- Cultures in reconstituted skimmed milk:
The milk was reconstituted at 100 g/l and pasteurized for 10 minutes at 90° C.
After around 2 hours of culture, a rise in pH in the culture of the strain
RD298 is observed (FIG. 1). The 6 spontaneous mutants have a very regular acidification
curve, with no pH rise nor temporary slowing down of the acidification speed. At
certain times in the culture, the shift in acidification compared with the parent
strain reaches almost 4 hours. This therefore allows a given value of pH to be
reached more quickly. The importance of this observation is major: if it is wished
to reach a given pH without reducing the incubation time, a ur(-) strain can be
used, reducing the amount of seeding compared with the amount used with a ur(+)
strain. Certain of the mutants obtained after treatment with NTG have a behaviour
similar to the spontaneous mutants; others acidity the medium more slowly (298-3.3)
or more quickly (298-10).
With the exception of the mutant 888-1 the ur(-) spontaneous mutants of ST888
have the same acidification curve. As for RD298, a more regular and faster acidification
is observed with the mutants (FIG. 2).
- Cultures in UHT sterilized skimmed milk (Lactel®):
As for the cultures carried out in reconstituted milk, a temporary halt in the
lowering of the pH is observed with the strain RD298, this phenomenon being absent
in the cultures of the spontaneous ur(-) mutants (FIG. 3).
The ur(-) mutants isolated from ST888, whether they are spontaneous or obtained
by treatment with NTG, have an acidification curve more regular than that of the
parent strain (FIG. 4).
- Effect of variations in the composition of the milk on the acidification curves:
The strain RD298, and the ur(-) mutants 298-K and 298-10, were cultured on UHT
sterilized skimmed milk supplemented or nor with different amounts of urea. The
initial urea concentration of the milk was equal to 3 mM and the urea contents
of the different cultures were contained within the variation zones that are usually
observed with cow's milk. It should be noted that, unlike the ur(-) mutants, the
acidification curves obtained with the parent strain are highly dependent on the
urea content of the milk (FIG. 5).
The authors of the present invention also observed that the acidification curves
obtained with the parent strain are dependent on the nickel and cobalt content
of the milk, which is not the case for the ur(-) mutants (FIG. 6).
In all the cultures described previously, it was observed that the strains RD298
and ST888 produced ammonia and hydrolyzed all the urea contained in the milk. No
ammonia production was observed with the mutants. This indicates that urea is the
main substrate used by
St. thermophilus for producing ammonia. Thus the
use of ur(-) strains makes it possible to avoid any ammonia production due to
St.
thermophilus during cheese manufacture. Consequently, the ammonia contents
of cheese-making wheys can be limited.
- Variability of the acidifying activities:
The authors of the present invention observed interestingly that the curves of
acidification in reconstituted skimmed milk obtained with a number of ur(-) mutant
strains had large variations compared with the curve obtained with their parent strain.
FIG. 7 thus shows the acidification curves for reconstituted skimmed milk, obtained
with the strain RD672, and with ur(-) mutants originating from this strain.
The strain RD672 is not very acidifying (solubilized soft cheese type technology).
The mutant 672-47(0) is distinctly more acidifying than the parent strain, while
the mutant 672-36(50) has a fairly similar acidification kinetic. The mutant 672-70
(0) is distinctly less acidifying than the parent strain and the mutant 672-24(50)
is a little less acidifying than the parent strain.
Example 4
Manufacture of "Solubilized Soft Cheese" Type Cheeses Using Either the
Ur(+) Industrial Strain Rd298 or the Ur(-) Mutant Strain 298-10 (a mutant of RD298).
a) General Points
Under the generic name cheese, there is found a very large number of products,
having a technology, a flora and organoleptic properties which are very diverse.
Technologically speaking, cheese results in the first place from
the coagulation of milk obtained by renneting, which will be followed by draining
of the coagulum thus obtained (mechanical operations such as cutting, stirring
and turning). During manufacture, the growth of the added ferments will cause a
lowering of the pH of the coagulum. The acidification kinetic (the change in pH
as a function of time) and the drainage kinetic condition the final composition
of the curds and therefore the intrinsic characteristics of the cheeses. This is
why, for a given technology, having control over the acidification and drainage
kinetics is essential.
b) Specific Features of the "Solubilized Soft Cheese" Technology Used.
The manufacture of "solubilized soft cheese" type cheeses corresponds to the
use of a technology with enzymatic dominance (important function of the rennet)
with specific manufacturing temperature profiles, such as that described in Table 7.
The conduct of the draining is characterised by:
- considerable acidification at the start of the method which conditions
the draining level. The acidification is provided by Streptococcus thermophilus:
the target pHs to be reached at the different manufacturing stages are summarized
in Table 7;
- fast removal of the whey increased by mechanical operations (cutting,
stirring and moulding of the coagulum);
- operations facilitating the removal of the whey (turning).
c) Monitoring of the Cheese Manufacturing
Table 7 summarises the different technological steps of the manufacturing carried
out and shows the process times which were necessary in each test to reach the
target pHs of each of these steps.
Two distinct milks were used, one containing less than 1 mM of urea and the other
5 mM of urea. The ferments used consisted either of the industrial strain RD298
known for its ability to hydrolyze urea, ur(+), or the strain 298-10, a spontaneous
mutant of this strain lacking this urea hydrolysis ability, ur(-).
Monitoring the acidification of the milk containing a very small amount
of urea (less than 1 mM) shows that the two strains used allow the target pHs of
each step to be reached in approximately identical times. Similarly, these objectives
are achieved with the ur(-) strain 298-10 when the manufacturing milk contains
significant amounts of urea (5 mM). On the contrary, in order to meet the target
manufacturing pHs with the strain RD298 in the milk containing 5 mm of urea, the
process times have had to be considerably lengthened.
This study therefore demonstrates the certain technological advantage of the
ur(-) mutant 298-10 compared with the ur(+) industrial mother strain RD298.
| TABLE 7 |
| Technological characteristics of a "solubilised soft cheese" type cheese manufacture |
| and technological description of manufacturing carried out with the ur(+) strain
RD298 or the |
| ur(-) strain 298-10 used as ferments from milk containing either 5 mM of urea
or less than 1 mM |
| of urea. |
| |
|
|
Process |
Actual process time (min) |
| |
Manufacturing |
|
time |
Milk with less than |
Milk containing 5 |
| Manufacturing |
temperature |
Target pH |
objectives |
1 mM of urea |
mM of urea |
| stage |
(° C.) |
(±0.05) |
(±10 min) |
RD298 |
298-10 |
RD298 |
298-10 |
| Milk |
38 ± 0.5 |
6.48 |
0 ± 10 |
0 |
0 |
0 |
0 |
| Renneting |
|
6.40 |
70 ± 10 |
70 |
60 |
100 |
60 |
| Moulding |
|
6.30 |
120 ± 10 |
120 |
110 |
140 |
110 |
| 1st turning |
35 ± 0.5 |
6.20 |
180 ± 10 |
190 |
170 |
280 |
170 |
| 2nd turning |
26 ± 0.5 |
5.50 |
300 ± 10 |
310 |
310 |
450 |
310 |
| 3rd turning |
20 ± 0.5 |
5.25 |
540 ± 10 |
540 |
530 |
700 |
530 |
BIBLIOGRAPHY
Juillard V., Desmazeaud M. J., Spinnier H. E. 1988. Revelation of a ureasic
activity in Streptococcus thermophilus. Canadian Journal of Microbiology.
34: 818-822.
Martin B., Coulon J. B., Chamba J. F., Bugaud C. 1997. Effect of milk urea
content on characteristics of matured Reblochon cheeses. Lait. 77: 505-514.
Spinnier H. E., Corrieu G. 1989. Automatic method to quantify starter activity
based on pH measurement. Journal of Dairy Research. 56: 755-764.
Terzaghi B. E., Sandine W. E. 1975. Improved medium for lactic streptococci
and their bacteriophages. Applied Microbiology. 29: 807-813.
Tinson W., Broome M. C., Hillier A. J., Jago G. R. 1982a. Metabolism of
Streptococcus thermophilus. 2. Production of CO2 and NH3 from urea. Australian
Journal of Dairy Technology. 37: 14-16.
Tinson W., Ratcliff M. F., Hillier A. J., Jago G. R. 1982b. Metabolism of
Streptococcus thermophilus. 3. Influence on the level of bacterial metabolites
in cheddar cheese. Australian Journal of Dairy Technology. 37: 17-21.
*
