Note: Descriptions are shown in the official language in which they were submitted.
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Fermented dairy product
The subject of the present invention is a new
process for the preparation of a fermented milk
composition, the said process making it possible to
obtain food compositions having a particularly high load
of lactic acid bacteria, even after packaging and storage
for prolonged periods.
State of the ark
Although lactic acid bacteria are generally
known to have beneficial effects on human health, only
certain categories of lactic acid bacteria are really
capable of adhering to the human intestinal cells, of
excluding pathogenic bacteria from human intestinal
cells, and/or of acting on the human immune system by
allowing it to react more strongly to external attacks.
Lactic acid bacteria are termed "probiotic" if they
possess at least one of these characteristics.
To date, relatively few lactic acid bacteria
are truly probiotic bacteria. For example, the strains
Lactobacillus casei ATCC53103, Lactobacillus acidophilus
CNCM I-1225, Bifidobacterium breve CNCM I-1226,
Bifidobacterium infantis CNCM I-1227 and Bifidobacterium
longum CNCM I-1228, have thus been scientifically
recognized as being probiotic bacteria since they are
capable of adhering to human intestinal cells, of
excluding pathogenic bacteria from human intestinal
cells, and of acting on the human immune system J. of
Dairy Science, ~$, 491-497, 1995; Applied Env. Microb.,
.a.2, 4121-4128, 1993) .
The probiotic lactic acid bacteria are often
also extremely sensitive to oxygen because of their
adaptation to the anaerobic living conditions found in
the intestinal tract. Furthermore, these bacteria grow
poorly in milk, which poses problems for obtaining a
sufficient level of lactic acid bacteria in a fermented
dairy product.
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To enhance the growth of slow-growing lactic
acid bacteria, EP154614 suggests increasing the load of
the starter culture during the inoculation of a milk, and
also adding to this milk growth activators such as yeast
extracts or whey proteins, for example.
Although it is possible to solve, by this
means, the problems of growth in milk, the probiotic
lactic acid bacteria unfortunately still remain very
sensitive to the conditions for the processing and
preservation of a fermented milk. Indeed, most of the
plastic packagings used to package dairy products are
permeable to oxygen. Furthermore, the subsequent
processing of a fermented milk, for example to fromage
blanc or to liquid acidified milks, requires stirring of
the milk in the presence of air, which increases the
level of oxygen in the final product.
The present invention aims to overcome the
disadvantages of the prior art by providing a process
which promotes the survival of lactic acid bacteria.
Summary of the invention
To this effect, the present invention relates
to a process for the manufacture of a fermented milk
composition in which a milk is heat-treated at a
temperature and for a period such that its redox
potential at 25°C is reduced to a value of less than 450
mvolt, and the milk is inoculated with lactic acid
bacteria.
The present invention also covers all the milk
compositions packaged in a material which is impermeable
or semipermeable to oxygen, the said compositions
comprising at least 106 cfu/ml of probiotic lactic acid
bacteria and a redox potential of less than 450 mvolt
(cfu comes from the expression "colony forming unit").
Likewise, the invention also relates to the use
of a milk having a redox potential at 25°C which is less
than 450 mvolt, for the preparation of a dairy product
comprising lactic acid bacteria.
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Finally, the invention also relates to the use
of a fermented dairy composition derived from the present
process in the preparation of a dairy product comprising
lactic acid bacteria.
S Against all expectations, the lactic acid
bacteria which are known to be sensitive to the
conditions for fermentation and preservation of a milk,
for example which are sensitive to the presence of air,
in fact become more resistant to these conditions, and in
particular become tolerant to the presence of air, as
long as the redox potential of the milk in which they
live is less than about 450 mvolt. This resistance
results in a better bacterial development during the
fermentation of a milk, and a better survival of the
bacteria during the preservation of the fermented milk.
Although this redox potential can be adjusted
by various means, it has been found that a prolonged
pasteurization of the milk is sufficient to obtain a
required redox potential. Indeed, a prolonged heat
treatment makes it possible to break certain milk
proteins and thus release reducing groups. Furthermore,
this treatment makes it possible to cause the proteins
and the milk sugars to react so that the reducing
compounds derived from Maillard reactions are formed.
This pasteurization has other advantages.
Firstly, a prolonged treatment of the milk at high
temperatures promotes degassing of the milk, and
therefore a low oxygen content in the milk. Secondly,
this treatment makes it possible to convert a portion of
the milk lactose to lactulose which is known to stimulate
the growth of certain lactic acid bacteria.
brief description of the figures
- Figure 1 represents, after fermentation, the
number of cells of the CNCM I-1225 strain (cfu/ml) having
grown in various milks, the said milks having undergone
various heat treatments before fermentation, the said
fermented milks having been stored for 1 or 28 days at
refrigeration temperatures, and the said fermented milks
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having also been packaged in glass or polystyrene
packagings.
- Figure 2 represents, after fermentation, the
number of cells of the CNCM I-1225 strain (cfu/ml) having
grown in various milks, the said milks having a redox
potential of the order of 500 mvolt or -50 mvolt before
fermentation, and the said fermentation having been
carried out under aerobic or anaerobic conditions.
- Figure 3 represents the number of cells of
the CNCM I-1225 strain (cfu/ml) having grown in various
milks, as well as the redox potential at 25°C of these
fermented milks, as a function of the duration of storage
of these fermented milks at refrigeration temperatures.
Detailed description of the invention
Within the framework of the present invention,
the name "rennet" is given to the coagulating extract
obtained from the abomasum of young ruminants slaughtered
before weaning. It will be assumed that rennet also
covers calf rennet substitutes such as animal pepsins;
the coagulating preparations obtained from the plant
kingdom extracted from artichoke, thistle, ficin, latex,
fig, papain, for example; the coagulating preparations
obtained from the microbial kingdom extracted from
bacteria of the genus Bacillus and Pseudomonas, and
moulds belonging to the species Endothia parasitica,
Mucor pusillus and Mucor miehei, for example.
Milk is intended to designate, on the one hand,
a milk of animal origin, such as cow's, goat's, sheep's,
buffalo's, zebra's, horse's, ass's and camel's milk and
the like. This milk may be a milk in the native state, a
reconstituted milk, a skimmed milk, or a milk
supplemented with compounds necessary for the growth of
the bacteria or for the treatment of the milk, such as
fats, yeast extract, peptone, ascorbic acid and/or a
surfactant, for example. Preferably, these milks have a
pH of the order of 6.4-7, in particular pH 6.6-6.8.
The term milk also applies to what is commonly
called a vegetable milk, that is to say an extract of
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plant materials, treated or otherwise, such as legumes
(soya bean, chickpea, lentil and the like) or oilseeds
(rapeseed, Soya bean, sesame, cotton and the like), which
extract contains proteins in solution or in colloidal
suspension, coagulable by chemical action, by acid
fermentation and/or by heat. It has been possible to
subject these vegetable milks to heat treatments similar
to those for animal milks. It has also been possible to
subject them to treatments which are specific to them,
such as decolorization, deodorization, and treatments for
eliminating undesirable tastes. Finally, the word milk
also designates mixtures of animal milks and of vegetable
milks. Preferably, these milks have a pH of the order of
6.4-7, in particular pH 6.6-6.8.
Against all expectations, it has been found
that the growth and the survival of certain lactic acid
bacteria are also influenced by the water activity of the
milk (Aw), that is to say the ratio between the partial
vapour pressure of the water at the surface of the powder
and the vapour pressure of pure water at the same
temperature. The best survival levels may be obtained
when the Aw of the milk at 20°C is greater than 0.97,
preferably between 0.988-0.983, for example. As a guide,
the Aw may be determined by measuring the equilibrium
relative humidity reached in a closed vessel at constant
temperature. For that, a sample of a few g of milk is
enclosed in a leaktight container placed in a
thermostatted chamber at 20°C. The empty space around
this sample reaches, at equilibrium, after 30-60 min, the
same Aw value as the sample. An electronic sensor,
mounted in the lid of the container, then measures the
humidity in this empty space by means of an electrolytic
resistance.
Likewise, it has been found that the addition,
to the milk, of at least one bacterial growth promoting
agent made it possible to substantially increase the
growth and survival of certain lactic acid bacteria.
Among these agents, there may be mentioned in particular
a sugar such as glucose and sucrose, an amino acid such
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as cysteine and glutathione, a yeast extract, in
particular an extract comprising large quantities of
purine and pyrimidine bases as well as their phosphate
derivatives (adenosine, thymine, guanine, cytosine and
uracil) and/or hydrolysates of animal or plant (soya)
protein materials, for example. In particular, the milk
may comprise about 0.1-1% of yeast extract and/or about
0.25-1% of peptones.
Because it has been realized that the redox
potential of a dairy product comprising lactic acid
bacteria is capable of being substantially increased
during the storage of the dairy product at refrigeration
temperatures, it is also preferable to add to the milk
compounds which are capable of stabilizing its redox
potential. Among these compounds, there may be mentioned
all dietary reducing agents such as ascorbic acid,
vitamin E and/or their derivatives, which can be used in
an amount of 0.01-1% by weight, for example.
The compounds which stabilize the redox
potential and the agents which promote bacterial growth
may be added to the milk before pasteurization. However,
because some of these compounds can be destroyed or even
modified following a prolonged heat treatment, it is also
possible to envisage adding them to the milk after
pasteurization and/or after fermentation, in the form of
a sterile solution, for example.
All the devices intended for pasteurizing a
milk may be used by persons skilled in the art. It is
thus possible to heat treat the milk at at least 90°C for
at least 30 min, preferably at 95-130°C for 30-120 min,
so as to obtain a redox potential of less than 450 mvolt,
in particular of less than 400 mvolt, or even of less
than 350 mvolt if it is desired to obtain maximum growth
and survival of the lactic acid bacteria, for example.
Next, the pasteurized milk is inoculated with
at least one strain of lactic acid bacteria so as to
directly obtain in the milk from 103 to 10$ cfu/ml. It is
possible to inoculate the milk with a fresh culture of
lactic acid bacteria, with a concentrated and frozen
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culture, or even with a culture dried by lyophilization
or by spraying under a stream of hot air, for example.
The strain of lactic acid bacteria may be
chosen from the species Lactococcus lactis in particular
L. lactis subsp. cremoris, L. lactis subsp. lactic biovar
diacetylactis, and L. Iactis; Streptococcus thermophilus;
the acidophilic bacteria comprising L. acidophilus, L.
crispatus, L. amylovorous, L. gallinarum, L. gasseri, L.
johnsonii; Lactobacillus fermentum; Lactobacillus casei
in particular L. casei subsp. casei; Lactobacillus
delbruckii in particular L. delbruckii subsp. lactis; L.
delbruckii subsp. hel veticus; L. delbruckii subsp.
bulgaricus; the bifidobacteria, in particular
Bifidobacterium infantis, Bifidobacterium breve;
Bifidobacterium longum; and finally Leuconostoc
mesenteroides in particular L. mesenteroides subsp.
cremoris, for example (Bergey's Manual of Systematic
Bacteriology, Vol. 2, 1986; Fujisawa et al., Int. Syst.
Bact, ~, 487-491, 1992).
Preferably, lactic acid bacteria are used which
are sensitive to oxygen, in particular all the
bifidobacteria, Lactobacillus acidophilus, Lactobacillus
johnsonii, Lactobacillus gasseri, Lactobacillus
fermentum, Lactobacillus casei, Lactobacillus bulgaricus
and Lactobacillus helveticus.
The probiotic bacteria are of special interest
within the framework of the present invention. These
bacteria are in fact capable of adhering to human
intestinal cells, of excluding pathogenic bacteria from
human intestinal cells, and of acting on the human immune
system by allowing it to react more strongly to external
attacks (immunomodulation capacity), for example by
increasing the phagocytosis capacities of the
granulocytes derived from human blood (J. of Dairy
Science, ~, 491-497, 1995: immunomodulation capacity of
the La-1 strain which has been deposited at the Pasteur
Institute under the number CNCM I-1225).
By way of example, it is possible to use the
Lactobacillus acidophilus CNCM I-1225 strain described in
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EP577904. This strain was recently reclassified among the
Lactobacillus johnsonii, following the new taxonomy,
proposed by Fujisawa et al., which is now authoritative
in the field of taxonomy of acidophilic lactobacilli
(Int. J. Syst. Bact., ~, 487-791, 1992).
The milk composition obtained by the process
according to the invention may also be traditionally
fermented until at least 106 cfu/ml, in particular 10'-109
cfu/ml, are obtained, for example. When these milk
compositions comprise probiotic lactic acid bacteria, in
particular the L. johnsonii CNCM I-1225 strain, it is
preferable to carry out the fermentation in the absence
of oxygen, for example under a carbon dioxide atmosphere.
The milk composition obtained by the process
according to the invention may also be converted to
unripened fromage frais which are commonly called "quarg"
or "cottage cheese" in Anglo-Saxon countries and "quark"
in Germany, for example. For that, it is possible to
ferment the milk inoculated with lactic acid bacteria,
but not necessarily. Rennet, of the order of 0.01 to
0.15% by volume/volume, is generally added to it so as to
cause the casein to pass from a colloidal phase to a
precipitated phase, this passage being accompanied by the
formation of a whey. Next, the whey is separated by
centrifugation or ultrafiltration.
The invention also covers all milk compositions
packaged in a material which is impermeable or
semipermeable to oxygen, the said compositions comprising
at least 106 cfu/ml of probiotic lactic acid bacteria and
a redox potential of less than 450 mvolt, preferably of
less than 400-350 mvolt if compositions are desired in
which the viability of the lactic acid bacteria is
stabilized at an acceptable level.
Preferably, the milk compositions according to
the invention are packaged in a material which allows the
passage of less than 0.01 cm3 of air per day and per cm2
under an external pressure of 0.21 bar, for example a
material which is impermeable to air such as glass or
ethyl vinyl alcohol (EVOH), or a material which is
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semipermeable to air such as polystyrene (PS),
polypropylene (PP), polyethylene terephthalate (PET),
ethyl vinyl alcohol (EVOH), high-density polyethylene
(HDPE), or a mixture of these materials, for example.
Because the lactic acid bacteria which exist in
a milk treated according to the present process become
particularly resistant to stress situations, the milk
composition obtained by the present process can also be
used to prepare other fermented dairy products, in
particular as a starter for a fermentation of a milk on a
large scale, for example.
The present invention is described in greater
detail below with the aid of the additional description
which follows, which refers to examples of preparation of
fermented dairy products, as well as to the description
of a test for measuring the redox potential. The
percentages and parts are given by weight unless
otherwise stated. It goes without saying, however, that
these examples are given by way of illustration of the
subject of the invention and do not constitute in any
manner a limitation thereto.
Measurement of the redox potential
The measurement of the redox potential is
carried out in accordance with the publication by Buhler
H. et al. (Ingold A.G., Germany). For that, a pH/mvolt-
meter combined with a redox electrode (Ingold No.
105053288) is used. The pH/mvolt-meter is calibrated
using a standard redox buffer. The milk samples at pH
6.4-7 are incubated beforehand on a bath at 25°C. The
measurement of the redox potential is carried out after 3
min of stability, and the redox potential is calculated
by adding 244.4 mvolt to the redox value displayed.
Example 1
Several milk samples consisting of 10% of a
skimmed milk powder, 1% of yeast extracts and 0.5% of
glucose are prepared. In order to obtain redox potentials
of less than 450 mvolt, these milks are heat-treated,
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respectively, for 30 min at 63°C on a hot water bath, for
30 min at 95°C on a hot water bath, for 15 min at 121°C
in an autoclave, or for 60 min at 121°C in an autoclave.
These milks are inoculated with the probiotic strain
Lactobacillus johnsonii CNCM I-1225 which was deposited
at the Pasteur Institute, 25 rue du docteur Roux, Paris,
30 June 1992. These milks are fermented without stirring
until a pH of the order of 4.6 is obtained, they are each
packaged in two impermeable (glass) or semipermeable
packagings, each packaged milk is stored for 1 day or 28
days at refrigeration temperatures, and then after
storage, the number of lactic acid bacteria which survive
is determined.
The results presented in Figure 1 clearly show
that the prolonged heat treatment of the milk
substantially enhances the survival of the lactic acid
bacteria, even in the presence of oxygen. Moreover, it is
also observed that the redox potentials of the heat-
treated milks are inversely correlated with time and with
the temperature level applied to the milk. In other
words, the more extensive the heat treatment of the milk,
the lower the redox potential of the milk. In this
regard, it should thus be noted that the lower the redox
potential of the milk, the more manifest the resistance
of the lactic acid bacteria.
The redox potentials at 25°C of two artificial
MRS media are adjusted respectively to about 500 mvolt
and -50 mvolt, by adding thereto an appropriate quantity
of potassium ferricyanide or of DTT. These two media are
inoculated with an inoculum of the Lactobacillus
johnsonii CNCM I-1225 strain, and they are fermented
under aerobic conditions. For the aerobic conditions,
sterile air bubbles are introduced into the fermentation
media. Finally, the number of bacterial colonies which
have grown in these milks is counted.
The results presented in Figure 2 show that the
milks having a redox potential at 25°C of the order of
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-50 mvolt give the best growth scores, whether in the
absence or in the presence of air. Consequently, when the
redox potential of a milk is reduced, the growth of
certain lactic acid bacteria is thereby promoted.
Two starters are prepared from the
Lactobacillus johnsonii CNCM I-1225 strain and from the
Streptococcus thermophilus CNCM I-1421 strain which was
deposited at the Pasteur Institute, 25 rue du docteur
Roux, Paris, 18 May 1994, in a milk consisting of 10% of
a skimmed milk powder, 1% of yeast extract and 0.5% of
glucose, the said milk having previously been heat-
treated at 95°C for 30 min.
A skimmed milk conventionally pasteurized at
115°C for 20 min is inoculated with 5% of the
Lactobacillus johnsonii CNCM I-1225 strain starter and
with 0.5% of the Streptococcus thermophilus CNCM I-1421
strain starter. When the pH of the fermented milks
reaches pH 4.5, 0.1% weight/volume of vitamin C is added,
the milks are packaged in pots which are semipermeable to
air and they are stored at refrigeration temperatures for
1, 14 or 28 days, after which the redox potential of the
fermented milks is measured and the number of
Lactobacillus johnsonii CNCM I-1225 bacteria which have
survived in these fermented milks is counted.
For comparison, a milk is fermented under the
same conditions, the only difference being that vitamin C
is not added.
The results presented in Figure 3 show that if
the redox potential of a fermented milk is reduced to and
stabilized at less than 450 mvolt, a survival rate of the
lactic acid bacteria of at least 50% is obtained after 28
days of storage; whereas if the redox potential of the
fermented milk is greater than 450 mvolt, less than 1%
survival is obtained after 28 days of storage.
Example 4
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Two milks consisting of 10% of a skimmed milk
powder and various concentrations of yeast extracts are
prepared, these media are heat-treated at 115°C for
15 min, they are inoculated with 5% of a fresh culture of
S the Lactobacillus johnsonii CNCM I-1225 strain, they are
incubated at 40°C for 1 to 28 days, and the number of
lactic acid bacteria which have survived these storage
conditions is determined.
For comparison, milks fermented and stored for
1 to 28 days are analysed, the said milks comprising no
yeast extracts.
The results show that the use of 0.1 to 1% of
yeast extracts in the culture medium promotes the
survival of the lactic acid bacteria during prolonged
storage of this medium. The best results are obtained for
milks having of the order of 1% of yeast extracts.
Several milks containing 10% of a skimmed milk
powder and various concentrations of additives are
prepared, these media are heat-treated at 115°C for 15
min, they are inoculated with 5% of a fresh culture of
the Lactobacillus johnsonii CNCM I-1225 strain, they are
incubated at 40°C until a pH of 4.5 is obtained, they are
cooled to 4°C for 28 days in pots which are permeable to
air or semipermeable to air, and the number of lactic
acid bacteria which have survived these storage
conditions is determined. The experimental conditions are
given in Table 1 below. It should however be noted that
vitamin C is added after fermentation and before storage,
in the form of a sterile solution.
The results presented in Table 1 below show
that the peptone extracts, the yeast extracts and/or the
vitamin C make it possible to enhance the survival of the
probiotic lactic acid bacteria after 28 days of storage
at refrigeration temperatures, even in the presence of
air.
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Media Packaging % survival
after 28
days
10% of a skimmed milk powder, permeable 0.22
1% of yeast extract, 0.5% of semipermeable14.03
glucose
10% of a skimmed milk powder, permeable 2.47
1% of yeast extract, 0.5% of semipermeable21.18
glucose, and 0.01% of vitamin
C
10% of a skimmed milk powder, permeable 0.34
1% of yeast extract, 0.25% of semipermeable10.55
peptone, and 0.01% of vitamin
C
10% of a skimmed milk powder, permeable 1.72
1% of yeast extract, 0.25% of semipermeable21.11
peptone, and 0.1% of vitamin
C
10% of a skimmed milk powder, permeable 28.09
1% of yeast extract, 0.25% of semipermeable32.85
peptone, and 0.5% of vitamin
C
10% of a skimmed milk powder, permeable 31.81
1% of yeast extract, 0.25% of semipermeable31.81
peptone, and 1% of vitamin C
Exam lp a 6
Several milks are prepared which consist of 10% of a
skimmed milk powder, 1% of yeast extracts and various
concentrations of sucrose so as to adjust the water
activity of the milk from 0.978 to 0.989. These media are
heat-treated at 115°C for 15 min, they are inoculated
with 5% of a fresh culture of the Lactobacillus johnsonii
CNCM I-1225 strain, they are incubated at 40°C until a pH
of 4.5 is obtained, they are cooled to 4°C, they are
stored at 4°C for 1 to 28 days in pots which are
semipermeable to air, and the number of lactic acid
bacteria which have survived these storage conditions is
determined.
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The results show that the lactic acid bacteria
survive storage better for a prolonged period at
refrigeration temperatures when the Aw of the culture
medium before fermentation is of the order of 0.985.
