Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
-- ~0 94/00019 213 8 ? 6 4 PCT/DK93/00207
BAKED PRODUCT CONTAINING VIABLE MICROORGANISMS AND PROCESS
FOR PREPARING SAME
FIELD OF INVENTION
The present invention provides baked products cont~'n'ng
dietetically desirable viable microorganisms and a process
for preparing such products.
TECHNICAL BACKGROUND
Under normal conditions, the gastrointestinal tract of ani-
mals including humans is colonized by a numerous and diverse
population of indigenous microorganisms. This naturally
occurring intestinal microflora plays a significant role in
maintaining the hosting macroorganism in a healthy condition
due to several ecological effects of this microflora. First
of all an intact intestinal flora will constitute a barrier
against colonization of pathogenic organisms passing the
gastrointestinal tract and thereby provide a natural protec-
tion against microbial diseases. Secondly, the indigenous
microflora produce a number of enzymes and other metabolites
which may supplement the digestion of nutrients which is
caused by the natural digestive enzymes of the macroorganism.
Under certain conditions, however, the number of these nat-
urally occurring intestinal microorganisms may be decreased
or the beneficial balance between the species hereof may
become less favourable to the host organism. Examples of such
conditions are stress conditions, treatments with anti-
microbial medicaments or inappropriate diets. These condi-
tions may result in an increased susceptibility to acquiring
diseases or in functional disturbances in the digestive
t~acts such as e.g. diarrhoea or constipation.
WO94/00019 PCT/DK93/00207
2~38764 2
~ Under such less favourable conditions it may be advantageous
to correct the disturbances of the natural microbial flora by
administering to an individual suffering from such disturb-
ances a large number of beneficial microorganisms which are
capable of surviving or even colonizing the gastrointestinal
tract. Examples of microorganisms which are currently admin-
istered with this purpose are lactic acid bacteria and yeast
cultures.
The ~flmin; stration of beneficial microorgAni ~m~ iS not only
considered advantageous for individuals having recognizable
disturbances of their gastrointestinal flora. It is thus
contemplated that even individuals without such recognizable
disturbances may benefit from the ~flm;n;stration as the
above-defined function of their indigenous flora may be
enhanced hereby.
~m; n; stration of dietetically effective microbial cultures
may be via the diet such as by consumption of fermented dairy
products or other food products including meat products and
fermented vegetable products, cont~in;ng viable lactic acid
bacteria. However, the cultures may also conveniently be
administered in the form of concentrates of the microorga-
nisms, e.g. in the form of suitably formulated preparations
including powders, granulates, tablets or capsules containing
a high number of one or more species of the beneficial micro-
organisms. In general, such concentrated dosage forms arerelatively expensive and hence, their distribution is still
limited.
From a dietetic point of view it is advantageous to adminis-
ter the beneficial microorganisms as a part of the normal
diet. However, many hnm~n~ dislike a constant intake of the
above-mentioned fermented food products. Therefore, it is
desirable to incorporate the beneficial microorganisms in
types of food products which are consumed universally and
regularly in considerable quantities by a majority of con-
,~094/000l9 X 13 8 7 6~gpcT/DKg3/oo2o7
sumers. In this respect, bread and other bakery productscomply with this requirement.
However, bakery products are usually subjected to baking
temperatures which will kill microorganisms except extremely
thermotolerant species or thermotolerant forms of microor-
ganisms. The present invention provides for the first time
baked products which contain a dietetically desirable number
of beneficial microorganisms as they have been defined above.
SUMMARY OF THE INVENTION
Accordingly, in one aspect the present invention relates to a
method of preparing a bakery product containing viable micro-
organisms, comprising the steps of (1) preparing a baked
product and cooling it to a temperature in the range of 0 to
70C, (2) preparing a suspension of microorganisms, contain-
ing 107 to 10l2 viable organisms per mL, (3) injecting into
the baked product a volume of the suspension of micro-
organisms which is in the range of 2 to 20 mL per kg of
product, to obtain the baked product having a content of
viable microorganisms which is in the range of 103 to
2 x lCl per g including contents which are in the range of
2 x 105 to 2 x 101 per g.
In an interesting embodiment of the invention there is also
provided a method wherein the suspension of microorganisms is
injected by means of an apparatus for injecting a suspension
of microorganisms into a baked product, comprising
(1) means for introducing the suspension into the baked
product,
(2) means for containing the suspension to be injected,
(~) means for connecting the reservoir with said means
for introducing the suspension,
WO94/00019 PCT/DK93/00207
2138764 4
(4) means for pneumatically transporting the suspension
from the containing means and to the introducing means,
and for pneumatically introducing the means for introduc-
ing the suspension into the baked product,
(5) means for adjusting the volume to be injected,
(6) means for maintaining the baked product to be
injected in a position which allows the introduction of
the suspension thereinto.
In another aspect, the present invention pertains to a baked
product having a content of viable microorganisms which is in
the range of 103 to 2 x l0l per g.
DETAILED DISCLOSURE OF THE INVENTION
Doughs or batters for several types of bakery products con-
tain viable microorganisms in large numbers. Thus, baker's
lS yeast is added during the preparation of the dough for many
types of wheat flour based bread types including rolls and
buns. In these types of bakery products baker's yeast is
added as a leavening agent and the leavening is a result of
the production of gas from the metabolically active yeast
cells.
In the preparation of certain bread products including rye-
bread, viable bacterial cultures such as cultures of lactic
acid bacteria are also added to the dough. The purpose hereof
is to confer to the bread a desired flavour of organic acids
and other aromatic compounds.
Traditionally, bread products in which wheat flour is par-
tially or completely replaced by rye flour, are prepared from
doughs comprising as a substantial ingredient a so-called
"leaven" which is a culture of one or several species of
,V094/00019 21 3 8 7 6 I PCT/DK93/00207
lactic acid bacteria grown in an aqueous suspension of flour.
The lactic acid bacterial culture in such leavens may ori-
ginate from the naturally occurring flora of lactic acid
bacteria of the flour, or suitable commercial lactic acid
bacterial starter cultures may be added. Suitable lactic acid
bacterial species for use in doughs include homofermentative
~ species which predom;n~ntly produce lactic acid when sugars
are fermented by them, and heterofermentative species which
when fermenting sugars, in addition to lactic acid produce
other acids including acetic acid and possibly propionic
acid.
When rye bread leavens are based on the propagation of indi-
genous lactic acid bacteria of the flour ingredient, a con-
tinuous process is normally used whereby a substantial amountof an outgrown leaven culture is used as an ingredient of a
new leaven mixture.
However, in all types of bakery products where microorganisms
are added during the preparation of the dough or batter,
these organisms are inevitably killed during the baking step
as a result of heat inactivation. Accordingly, fresh baked
product do not normally contain viable microorganisms.
As it has been mentioned above, the present invention pro-
vides a method of preparing a bakery product containing a
high number of dietetically desirable viable microorganisms
which are introduced into the baked product by injecting into
the baked product a suspension of the microorganisms.
As it is essential that the bakery product environment into
which the live microorganisms are introduced allows the
organisms to survive, the products should be cooled to a
temperature which is not detrimental to the viability of the
used microorganisms, prior to the introduction hereof. The
thermotolerance, i.e. the highest temperature at which sub-
stantially none of the cells of a suspension of microorga-
nisms as used herein are injured to an extent where they are
WO94/00019 PCT/DK93/00207
2i38~ 6 4 6
no longer capable of growing under suitable growth condi-
tions, may vary considerably.
In general, a non-injuring temperature as defined above is in
the range of 0 to 70C. Accordingly, the baked product is
cooled to a temperature within this range, the required
cooling depending on the microorganism(s) used. Typically,
the product is cooled to a temperature in the range of 20 to
65C such as in the range of 50 to 60C. In this context, it
is important to note that a temperature gradient will occur
during the cooling of a baked product. Depending on the
desired site of injecting the microbial suspension the cool-
ing is continued until a suitable temperature as defined
above has been reached at that particular site.
In accordance with the present invention, a suitable micro-
organism is one which confers to the baked product a desired
dietetically beneficial effect as it has been explained above
and which microorganism is capable of maint~; n ing its viabil-
ity after injection into the product to a degree which
results in the presence of a dietetically effective amount of
viable organisms herein at the time of consumption of the
baked product. Such a useful microorganism may be selected
from a fungal species, a yeast species and a bacterial spe-
cies.
One obvious prerequisite for obt~ining the dietetically
beneficial effect is to select microorganisms which when
present in a baked product is capable of resisting the acidic
conditions in the anterior parts of the gastrointestinal
tract, in particular in the stomach and the anterior parts of
the small intestines. Accordingly, particularly interesting
microorganisms are microorganisms which inherently are tole-
rant to acidic conditions. However, it is also possible to
use less acid tolerant organisms provided they are provided
in a form where they are protected against gastrointestinal
acidic conditions. Thus, the microorganism(s) may be coated
or encapsulated by a suitable compound or suitable compounds
213876~~094/00019 PCT/DK93/00207
7 -
~
which are insoluble under acidic conditions such as in thestomach or the anterior parts of the small intestines but
which is/are dissolved when present in an environment with a
pH in the range of e.g. 5 to 10. Any suitable coating or
encapsulation method known in the art may be used.
In accordance with the invention, the suspension of micro-
organisms being injected may comprise organisms belonging to
the same species or it may comprise microorganisms which are
a mixture of species of microorganisms. In advantageous
embodiments of the invention, the microorganisms are orga-
nisms belonging to one or more species of lactic acid
bacteria. This group of microorganisms having as a common
characteristic the ability of producing lactic acid bacteria
under microaerophilic or anaerobic conditions, comprise
several genera including gram-positive organisms such as
Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and
Pediococcus and Bifidobacterium. The genus Lactobacillus
include as typical examples the following species
Lactobacillus acidophilus, Lactobacillus plantarum, Lacto-
bacillus casei, Lactobacillus delbruckii, and Lactobacillushilgardii. A typical Bifidobacterium species is Bifidobacte-
rium bifidum. Among Streptococcus spp. Streptococcus thermo-
philus and Streptococcus faecium may be used in accordance
with the present invention.
In addition to bacteria belonging to the above group of
lactic acid bacteria certain Propionibacterium spp which are
gram-positive anaerobic bacteria capable of fermenting lac-
tate to propionate, including Propionibacterium shermanii may
be used, optionally in combination with one or more lactic
acid bacterial species such as e.g. Lactobacillus acidophi-
lus, Bifidobacterium bifidum or Streptococcus faecium.
Several lactic acid bacterial species are com~onl y used as
starter cultures in the manufacturing of fermented food
products, such as dairy products, meat products, vegetable
products and as mentioned above, in the manufacturing of
WO94/0~l9 PCT/DK93/00207
~,~3~'1G4 8
bread products. Frequently, such lactic acid bacterial -
starter cultures comprise a mixture of species. One typical .
example hereof is the use of a mixture of Lactobacillus
acidophilus and Bifidobacterium bifidum in certain fermented
milk products.
From a dietetic point of view, the use of a mixed culture of
these species may be advantageous as Lactobacillus acidophi-
lus is considered to be particularly adapted to remain viable
or even colonize in the anterior parts of the
gastrointestinal tract whereas Bifidobacterium bifidum which
is a strictly anaerobic organism assumingly will colonize the
posterior parts of the intestines where the Eh is low. It is
contemplated that a dietetically beneficial effect may be
obtained throughout the major part of the gastrointestinal
tract by providing in a food product including the baked
product according to the present invention, a mixture of
lactic acid bacterial species each of which is particularly
adapted to colonize in different parts of the
gastrointestinal tract.
Accordingly, in one interesting embodiment of the present
invention a useful suspension of microorg~n~sm~ comprises a
mixture of a mlcroaerophilic lactic acid bacterium such as a
Lactobacillus species and a strictly anaerobic lactic acid
bacterial species including a Bifidobacterium species.
However, under certain conditions it may be desirable to use
only one species.
As it has been mentioned above, useful microorganisms may in
the present context include yeast organisms. Typical examples
hereof are Saccharomyces cerevisiae e.g. in the form of
baker's yeast, and Klyveromyces lactis. In accordance with
the invention, the culture of microorganisms to be injected
into the bakery products may comprise a mixture of bacterial
and yeast organisms.
, ~l3876~
~094/00019 PCT/DK93/00207
In accordance with the invention, the microorganisms may be
wild-type strains as isolated from their natural environment,
It may, however, be advantageous to use microbial strains
which have been improved be selection, by mutation or by
genetic recombination.
From a hygienic point of view it is important that the
injected suspension of microorganisms is biologically pure,
i.e. it should only contain the desired microorganisms and no
or only few foreign microorganisms as cont~mln~ting orga-
nisms. In bakery products contamination with undesired fungiare particularly serious as these organisms may be capable of
multiplying under the conditions prevailing in bakery pro-
ducts including the low aw. Accordingly, the term "suspension
of microorganisms" as used herein denotes a substantially
pure suspension of the desired microorganisms.
The suspension of microorganisms being injected suitably
contains 107 to 10l2 viable org~nism~ per mL. In certain
preferred embodiments, the content of microorg~nlsmc is in
the range of 109 to 101l per mL. Conveniently, the suspension
of microorganisms is prepared from a concentrate of organisms
cont~lnlng lo8 to lol3 organisms per g. Such concentrates may
be in the form of a slurry or paste of freshly grown micro-
bial cells. However, in industrial production it may be more
convenient to prepare the suspension from a frozen or freeze-
dried concentrate of the microorganism(s) optionally contain-
ing one or more cryoprotective substances.
The suspension of microorganisms may e.g. be prepared from a
concentrate of organisms as defined above, by suspending 1
part of the concentrate of microorganisms in 5 to 20 parts of
an aqueous medium comprising at least one microbial nutrient
and at least one salt. In the present context, an aqueous
medium may be one selected from distilled water, deionized
water or tap water. It is well-known that in a concentrate of
mic-oorganisms as in the one presently defined, a certain
proportion of the organisms may be in state of "stress" or
WO94/00019 ~ 6 4 lo PCT/DK93/00207
sublethal injury, where maintenance of their viability is
dependent on the presence in their envi~ol~lle~t of certain
nutrients.
Accordingly, the use of such sublethally injured microor-
ganisms in the present invention may require the addition to
the aqueous suspending medium of one or more nutrients which
cannot be synthesized or utilized by them. In this context,
suitable nutrients may be any nutrient normally used in
culturing media for particular microorganisms or the suspend-
ing medium may be a commercial liquid culture medium such ase.g. the conventionally used tryptic soy broth medium, con-
taining the required nutrients. Typically, such nutrients may
be selected from yeast extract which i.a. contain a variety
of vitamins, peptides and amino acids; a carbon source which
e.g. may be selected from a monosaccharide, a disaccharide or
a polysaccharide; and a vitamin or a mixture of VitAmi nc,
Furthermore, the suspension medium may suitably contain a
salt or a mixture of salts. The salt(s) may be selected from
an alkali metal salt such as NaCl or a phosphate and an
alkaline earth metal salts including phosphates, chlorides or
carbonates.
The suspension medium is preferably a sterile medium which
may be provided by using only sterile ingredients or by
subjecting the prepared medium to a treatment whereby con-
t~min~ting microorganisms are killed or removed. Such treat-
ments include heating at a temperature and for a period of
time which results in a sterile or substantially sterile
medium, and a filtering under conditions where microorganisms
are separated from the medium.
Subsequent to the preparation of the suspension of micro-
organisms, the suspension may be left to stand at a tempe-
rature in the range of 0 to 40C such as at ambient tempe-
rature for up till 6 hours such as about 15 to 120 minutes.
~094/000l9 769 PCT/DK93/002~7
The volume of the suspension of microorganisms typically
being injected into the baked product is in the range of 1 to.
50 mL, preferably in the range of 2 to 20 mL, per kg of
product. The appropriate volume depends on the type of bakery
product. It is essential that the volume injected does not
exceed the volume which can be absorbed by the product with-
out conferring to the product undesirable wet and "sticky"
spots which can be recognized by the consumer. It has thus
been found that the introduction into ryebread loaf products
of 3 to 15 mL per kg of product is suitable, provided this
volume is distributed by multiple injections such as by means
of a multiplicity of needles as it will be explained below.
In particular, a volume which is in the range of 5 to 12 mL
per kg such as 10 mL has proven to result in ryebread where
the injected suspension is appropriately absorbed into the
bread crumb.
The above volume ranges may be suitable for pieces of bakery
products which have a weight being in the range of 0.2 to 2
kg. When the bakery products to be injected are in smaller
pieces, such as buns or rolls having a weight e.g. in the
range of 0.02 to 0.2 kg the volumes may conveniently be in
the range of 0.1 to 2 mL, such as 0.25 to 1 mL.
In accordance with the invention, any bakery product having a
crumb structure and consistency allowing the injection and
absorption of the suspension of microorganism(s) may be
subjected to the injection of such a suspension. Interesting
bakery products include ryebread products, yeast-leavened
bakery products and chemically leavened bakery products.
Yeast-leavened bakery products include wheat flour based
bread products in the form of wheat flour-containing loaves,
buns or rolls, and pastry products. Among chemically leavened
bakery products typical examples are cakes such as sponge
cakes, pound cakes, scones and muffins.
WO94/~l9 PCT/DK93/00207 `~
~3 a~ 6 4 12
The bakery products containing the viable microorganisms may
be packaged for display in retail shops e.g. in air-tight
polymer foil materials, optionally as evacuated packages.
When the bakery product is one which is consumed in slices,
the packaged product may be provided as slices.
In one preferred embodiment of the present invention, the
suspension of microorganisms is injected into the baked and
cooled product through a multiplicity of needles having a
length which is in the range of 5 to 100 cm and a diameter
which is typically in the range of 0.5 to 3.0 mm such as
about 2 mm, said needles being connected to one or more
reservoirs contA;n;ng the microbial suspension(s). A suitable
length of the needles depend on the ~;m~n~ion of the product
to be injected. In useful embodiments the needle length is 10
to 40 cm.
The injection of the suspension of microorganisms into the
bakery product may take place during introduction of the
needles or it occur during the retrograde movements of the
needles. Most conveniently, however, the injection may take
place during introduction as this allows the inner space of
the needles to become refilled with the suspension during the
retrograde movement. The direction of the needles in relation
to the bakery product to be injected may be varied e.g.
depending on the outer ~;m~n~ions of the product. As an
example, a loaf of ryebread or a loaf of wheat flour bread
may conveniently be injected longitudinally.
Although the injection of the microbial suspension may be
carried out mAnllA1ly such as by a syringe or a multiplicity
of syringes provided with a needle, the injection is pre-
ferably carried out by means of an apparatus comprising
(1) means for introducing the suspension into the bakedproduct, (2) means for containing the suspension to be
injected, (3) means for connecting the reservoir with said
means fcr introducing the suspension, (4) means for pneumati-
cally transporting the suspension from the containing means
~ ~138764
WO94/00019 PCT/DK93/00207
13
and to the introducing means, and for pneumatically introduc-
ing the means for introducing the suspension into the baked -
product, (5) means for adjusting the volume to be injected,
and (6) means for maintA; n; ng the baked product to be
injected in a position which allows the introduction of the
suspension thereinto.
Conveniently, the means for introducing the suspension of
microorganisms into the baked product comprise a multiplicity
of needles having the above-defined ~;menRions. A suitable
number of needles depends on the ~lmenRion of the baked
product to be injected, a suitable number of needles typical-
ly being in the range of 2 to 20 such as e.g. in the range of
3 to 10. In certain useful embodiments, the needles are
placed equidistantly.
Suitable means for containing the suspension(s) of micro-
organisms include metal, glass and plastic containers such as
tanks, bottles or jars. When it is preferred to inject two or
more different suspensions separately, the means may comprise
a multiplicity of containers connected to different needles
thus allowing simultaneous injection of separate microbial
cultures. In advantageous embodiments, the means for pneu-
matically transporting the suspension of microorganism(s) is
activated during the introduction into the baked product of
the means for introducing the suspension.
As it is mentioned above, the suspension of microorganisms
may advantageously be injected into the baked product during
introduction of the needles. Accordingly, the apparatus may
be constructed so that the means for introducing the suspen-
sion is filled or refilled with suspension during retrograde
movements of the means.
In accordance with the invention, the means for adjusting the
total volume to be injected into one piece of bakery product
may be constructed so as tc allow adjustment cf a volume
which is in the range of 2 to 20 mL, preferably in the range
WO94/00019 PCT/DK93/00207
7 6 ~ 14
of 3 to 15 mL such as in the range of 5 to 12 mL, e.g. a
volume of about 10 mL. These volume ranges may be suitable
for pieces of bakery products which have a weight being in
the range of 0.2 to 2 kg. When the bakery products to be
injected are in smaller pieces, such as buns or rolls having
a weight e.g. in the range of 0.02 to 0.2 kg the volumes may
conveniently be in the range of 0.1 to 2 mL, such as 0.25 to
1 mL.
As mentioned above, the present invention provides in a
further aspect a baked product prepared by the method as
described herein which product has a content of viable
dietetically beneficial microorganisms as defined above,
being in the range of 103 to 2 x 101 per g. In preferred
embodiments the content of microorganisms may suitably be in
the range of 2 x 105 to 2 x 101 per g including contents in
the range of 2 x 104 to 2 x 109 per g.
The desired content of viable microorganisms depends on the
selected type(s) of microorganisms but it should preferably
be at a level where a dietetically effective amount of micro-
organicms is provided in a typical serving of the particularbakery product. As one example, about 100 g of bread may
typically be served for a meal. Assuming that a dietetically
beneficial amount of injected viable microorganisms per meal
is about lx108, the bread served should contain at least lo6
viable microorganisms per g. Accordingly, the injected amount
of microorg~nlsm~ must be adjusted to result in such a
desired mlnlmllm amount of viable microorganisms at any point
of time between manufacturing and consumption.
Most microorganisms which may be used in accordance with the
invention will not multiply during the above-defined period
of time but their number may gradually be reduced. According-
ly, the initial amount of viable microorganisms, i.e. the
amount hereof present in the baked product immediately fol-
lowing injection must be selected at a higher level than the
desired beneficial amount at the time of consumption so as to
2138764
~094/00019 PCT/DK93/00207
compensate for a possible reduction of viability during
storage. Typically, the desired initial amount of viable
microorganisms may be 5 to 100 times higher than the amount
required to provide the dietetically effective amount at the
latest possible time of consumption. Thus, as an example, a
loaf of ryebread may therefore have an initial content of
viable microorganisms which in the range of 5 x 108 to 109
per g.
As it has been mentioned above, the number of viable micro-
organisms in the subject baked product is defined as the
number hereof capable of growing under optimal conditions of
growth. The appropriate method of enumeration will depend on
the particular type of microorganism(s). Typically, such
methods may be conventional plate count methods using a
growth medium suitable for the microorganisms used and incu-
bating the medium inoculated with suitably diluted samples of
the product under suitable atmospheric and temperature condi-
tions and counting the number of colony forming units.
The invention is further illustrated in the following
examples:
EXAMPLE 1
Ryebread with Lactobacillus acidophilus and Bifidobacterium
bifidum
1. Preparation of "leaven mixture~ ingredient for ryebread
Initially, a "mother leaven" is prepared by inoculating 10 g
of a commercial culture of Lactobacillus acidophilus sus-
pended in 200 mL of demineralized water into a mixture of 2
kg of coarse rye flour and 2 L of water and incubating the
resulting mixture at 38C for about 8 hours followed by the
addition to the mixture of additional rye flour and water and
continued incubation at 38C. This is repeated on the 2nd day
WO94/00019 PCT/DK93/00207
~ on the 3rd day after which the leaven is ready for use in
bread manufacturing.
2. Manufacturing of ryebread loave~
A dough was prepared according to the following recipe:
Linseed 4.0 kg
Water for soaking the linseed 8.0 kg
Soybean shells 4.0 kg
Coarse rye flour 28.0 kg
Liquid leaven mixture 20.0 kg
10 Syrup 0.6 kg
Salt 0.6 kg
Breadcrumbs 1.6 kg
Risofarin~ 1) 0.6 kg
Water 22.0 kg
15 Baker's yeast 0.6 kg
1) A mixture of modified maize, rice and wheat starches
The ingredients except the soybean shells were mixed into a
dough by kneading in a kneading apparatus for about 15 minu-
tes and left to stand for about 15 minutes at a temperature
of about 28 to 30C.
In a subsequent step, the dough was portioned into 1100 g
units being formed into loaf shape and covered with soybean
shells and transferred to baking tins. The loaves were next
left to rise for about 50 minutes before being transferred to
an oven having an initial temperature of 270C and into which
steam was lead during the baking process. The baking time was
about 75 minutes during which the temperature was gradually
lowered to about 170C.
After the baking step, the loaves were taken to a cooling
tower in which air at a temperature of 5 to 7C was circu-
lated at a rate of 10.000 to 16.000 m3 per hour. After cool-
~094/00019 2 1 3 8 7 6 4 PCT/DK93/00207
17
ing for about 45 minutes, the temperature in the bread crumbwas about 66C.
3. Injecting a suspension of a mixture of Lactobac~llus
acidophilus and Bifidobacterium acidophilus
A suspension of microorganisms was prepared by suspending 1 g
of a frozen culture of Lactobacillus acidophilus (Nu-trish~,
Chr. Hansen's Laboratorium Danmark A/S, H0rsholm, Denmark)
containing about 5x101l viable organisms, 1 g of a frozen
culture of Bifidobacterium bifidum (Nu-trish~, Chr. Hansen's
Laboratorium Danmark A/S, H0rsholm, Denmark) containing about
8.3x1011 viable organisms in 10 g of ~uspension medium com-
prising 1 wt~ of sterile yeast extract, 0.9 wt~ of sterile
NaCl and 98.1 wt~ of sterile distilled water.
The resulting suspension was left to stand for up till 6
hours at a temperature of about 37C prior to injecting about
10 mL per kg into the baked and cooled loaves by means of a
multiplicity of needles having a length of about 40 cm and
diameter of about 3 m~..
4. Number of surviv~ng bacteria during storage of ryebread
loaves at 20-25C
The number of viable Lactobacillus acidophilus and Bifido-
bacterium bifidum was followed during storage at intervals of
2 days. Slices of the bread was homogenized in an aqueous
medium and serial dilutions hereof was plated onto MRS medium
(Oxoid CM561) and the plates incubated under anaerobic condi-
tions at 37C for about 3 days followed by counting the
number of colony forming units (CFU) per g of sample. The
initial counts of the two lactic acid bacteria were about
109 per g. The counts were reduced by 1-2 log units during
the following two days and kept stable at about 107 per g for
the following 8 days.
WO94/0~19 PCT/DK93/00207
- 2138~ 6 ~ 18
5. Qual~ty asseR~ment of the in~ected ryebread loaves
The injected loaves were assessed with regard to crumb struc-
ture, sensoric characteristics and crumb consistency. The
crumb structure showed an even satisfactory pore structure.
The bread had a rich smell of ryebread and a strong and rich
taste of rye and linseed. The salt and leaven contributed to
the overall desired taste but these ingredients did not
dominate the taste.
The consistency was assessed to be soft and suitably "wet"
and the bread did not stick to the knife when cut. This
desired consistency was kept satisfactorily during the 10
days period. The bread also had a desirable "resistance" on
chewing.
It was concluded that the injected loaves had sensoric and
consistency characteristics which are typical for ryebread
loaves of good quality.
EXAMPLE 2
Yeast-leavened retzel bakery product ("krinqle") containing a
mixture of Lactobacillus acidophilus. Bifidobacterium bifi-
dum, Streptococcus faecium and Propionibacterium shermanii
The retzel product was made from a dough contAinlng thefollowing ingredients: wheat flour, vegetable and ~n;mAl fat,
skimmed milk, eggs, baker's yeast, sugar, salt and wheat
starch. The following food additives were added: modified
starch, emulsifying agents (E 322, E 471, E 475, E 472e),
ascorbic acid, sodium carbonate, citric acid and flavouring
agent.
The product contained a filling comprising: AnlmAl and ve-
getable fat, sugar, muscovado, raisins, apricot kernels,
,~094/000l9 21 3 8 7 6 ~ PCT/DK93/00207
19
soybean flour, acidified skimmed milk, starch, water, salt,
egg white and whey powder.
Into 900 g of the baked and cooled product was injected
- 0.25 mL of separate suspensions cont~; nl ng Lactobacillus
acidophilus, Bifidobacterium bifidum, Streptococcus faecium
and Propionibacterium shermanii, respectively
The suspension medium for the bacterial cultures was Tryptone
Soy Broth (TSj3) (Oxoid CM129) and each of the bacterial
suspensions contained 1 g of bacterial cultures in 10 mL of
the suspension medium. The Lactobacillus acidophilus culture
used was the commercial frozen DVS ~direct vat set) culture
L. acidophilus La-5 (Chr. Hansen's Laboratorium Danmark A/S,
H0rsholm, Denmark) which contains at least 6 x 1ol0 colony
forming units (CFUs) per g. The Bifidobacterium bifidum
'5 culture was the frozen DVS culture Bifidobacterium Bb-12
(Chr. Hansen's Laboratorium n~nmArk A/S, H0rsholm, Denmark)
cont~'n;ng at least 1011 CFUs per g. The Streptococcus fae-
cium culture which was used for preparing a suspension was
the freeze-dried DRI-VAC culture Streptococcus faecium CH-1
(Chr. Hansen's Laboratorium Danmark A/S, H0rsholm, Denmark)
cont~lnlng 5 x 108 CFUs per g and the Propionibacterium
shermanii culture used was the frozen DVS Propionic Acid
Culture PS-1 (Chr. Hansen's Laboratorium Danmark A/S, H0rs-
holm, Denmark) containing 3 x 109 per g.
Subsequent to the suspending of the cultures the suspensions
were left to stand for resuscitation at 37C for 1-2 hours
and then injected into the retzel product at amounts of
0.25 mL of each suspension by means of a syringe and the
injected product was kept at room temperature until determi-
nation of viable bacterial counts.
About 24 hours after the injection of the bacterial suspen-
sions the contents of viable bacteria in a slice of about 1.5
cm thickness cut around the site of injection were determined
according to the following methods:
WO94/0~19 PCT/DK93/00207
21387 6 4 20
About 10 g of the above slice of product was homogenized in
saline in a Stomacher for about 4 minutes and appropriate
tenfold serial dilution were made. The viable counts of
Lactobacillus acidophilus and of Bifidobacterium bifidum were
done essentially according to the method defined in Example
1. The viable count of Streptococcus faecium was done by
plating onto blood agar (Blood agar base, Oxoid CM55 supple-
mented with 5~ calf blood) followed by aerobic incubation for
24 hours at 37C. The viable count of Propionibacterium
shPrm~nii cells was done by plating onto the above-defined
blood agar and incubating anaerobically at 37C for 48 hours.
The numbers of viable bacteria per g of the retzel product
were as follows:
(i) Lactobacillus acidophilus 4 x 107
15 (ii) Bifidobacterium bifidum 4 x 107
(iii) Streptococcus faecium 1 x 107
(iv) Propionibacterium shermanii 3 x 107
EXAMPLE 3
A sponqe cake ("sandka~e") containing a mixture of
Lactobacillus acidophilus. Bifidobacterium bifidum, Strepto-
coccus faecium and Propionibacterium shermanii
The sponge cake having a total weight of 350 g contained the
following ingredients: margarine, water, sugar, wheat flour,
starch, egg powder and milk powder. The following food addi-
tives were added to the batter: leavening agents (diphosphatesalt, sodium hydrogen carbonate), emulsifying agents (E 471,
E 322, E 475) and aroma.
0.25 mL volumes of the suspensions as defined in Example 2
were injected as also defined in Example 2, and after about
24 hours the bacterial counts (CFUs per g) were determined
.~094/00019 21 3 8 7 6 4 PCT/DK93/00207
21
according to methods defined in Example 2 with the following
results:
(i) Lactobacillus acidophilus 2 x 103
~ (ii) Bifidobacterium bifidum 1.2 x 105
5 (iii) Streptococcus faecium 1 x 107
~ (iv) Propionibacterium sherm3nii 2 x 103
EXAMPLE 4
White bread ("franskbr0d") containing a mixture of
Lactobacillus acidophilus. Bifidobacterium bifidum Strepto-
coccus faecium and Pro~ionibacterium shermanii
The bread was prepared from a dough cont~lning the followingingredients: Wheat flour, water, baker's yeast, ~nlm~l and
vegetable fat, caseinate, salt, sugar and glucose; and the
following food additives: emulsifying agents (E 482, E 475, E
471, E 322) and ascorbic acid. After baking and cooling to a
temperature below 70C, 0.25 mL of the bacterial suspensions
as defined in Example 2 were injected into a loaf of the
bread having a weight of 300 g and the viable counts deter-
mined after about 24 hours according to the methods as also
defined in Example 2. The results of these counts are shown
below:
(i) Lactobacillus acidophilus 4.3 x 105
(ii) Bifidobacterium bifidum 4 x 107
(iii) Streptococcus faecium 1 x 107
25 (iv) Propionibacterium shermanii 3 x 1 o7
WO94/000l9 PCT/DK93/00207
2~3~i 6 4 22
EX~MPLE 5
Whole-meal white bread ("4-kornsbr0d") containinq a mixture
of Lactobacillus acidophilus. Bifidobacterium bifidum. Strep-
tococcus faecium and Propionibacterium`shermanii
The bread was prepared from a dough consisting of wheat
flour, water, coarsely comm;nllted rye grains, whole-meal
wheat flour, baker's yeast, linseed, sesame seed, salt,
sugar; and the following food additive ingredients: emulsify-
ing agent (E 472e) and ascorbic acid. The dough was formed
into loaves each having a weight of 520 g.
After baking and cooling to a temperature below 70C, 0.25 mL
of the bacterial suspensions as defined in Example 2 were
injected into a loaf of the bread and the viable counts
determined after about 24 hours according to the methods as
also defined in Example 2. The results of these counts are
shown below:
(i) Lactobacillus acidophilus 2.4 x 106
(ii) Bifidobacterium bifidum 5 x 107
(iii) Streptococcus faecium 1 x 107
20 (iv) Propionibacterium shermanii 3 x 107
EXAMPLE 6
Rolls ("rundstykker") containing a mixture of Lactobacillus
acidophilus. Bifidobacterium bifidum. and Propionibacterium
shermanii
The rolls were prepared from a dough consisting of wheat
flour, water, baker's yeast, salt, sugar and eggs and a food
additive ingredient mixture, Compound 80 (Fællesforeningen
for Danmarks Brugsforeninger, Albertslund, Denmark). The
dough was formed into rolls each weighing about 3 0 g and
baked at 250 to 240C for about 12 minutes.
~094/~019 21 3 8 7 6 ~ PCT/DK93/00207
23
After baking and cooling to a temperature below 70C, 0.25 mL
of the bacterial suspensions as defined in Example 2 were
injected into each roll and the viable counts of the injected
bacteria were determined after about 24 hours according to
- 5 the methods as also defined in Example 2 except that a whole
roll of about 30 g was used the sample and homogenized by
means of the Stomacher. The results of these counts are shown
below:
(i) Lactobacillus acidophilus 4.5 x 105
(ii) Bifidobacterium bifidum 7.4 x 107
(iii) Propionibacterium shPrm~nii 2 x 108
EXAMPLE 7
Whole-meal white bread ("grovbr0d") containing a mixture of
Lactobacillus acidophilus, Bifidobacterium bifidum. and
Propionibacterium shermanii
This bread was prepared from a dough consisting of wheat
flour, water, whole-meal wheat flour, wheat grains, whole-
meal rye flour, baker~s yeast, ~n;m~l and vegetable fat,
sugar, salt and starch; and the following food additives:
emulsifying agents (E 471, E 472, E 322), phosphate (E 341)
and ascorbic acid. The dough was formed into loaves of a
weight of 450 g.
After baking and cooling the loaves to a temperature below
70C, 0.25 mL of the bacterial suspensions as defined in
Example 2 were injected into the bread and the viable counts
of the injected bacteria were determined after about 24 hours
and again after about 72 hours according to the methods as
also defined in Example 2. The results of these counts are
shown below:
WO94/~019 PCT/DK93/00207
2~3a~ 64 24
24 hours 72 hours
(i) Lactobacillus acidophilus 4 x lo6 3.8 x 106
(ii) Bifidobacterium bifidum 6.5 x 107 3.2 x 105
(iii) Propioni~acterium ~ 2.2 x 108 2 x lo8
~h~rrn;~ n i i
EXAMPLE 8
Dietary fiber-enriched rye bread containinq viable yeast
This bread was prepared from a dough cont~lnlng the following
ingredients: leaven (water, whole-meal flour of rye, leaven
culture), water, whole-meal flour of rye, whole rye grains,
dried crumbs of rye bread, salt, dried leaven (wheat flour,
wheat bran, water, leaven culture), wheat starch, yeast, malt
extract, syrup. The dough was formed into loaves of 1600 g,
baked and cooled to a temperature less than 70C.
15 A suspension of baker's yeast (Saccharomyces cerevisiae) was
prepared in the following manner: 1 g of the baker's yeast
(Malteserkorsgær~, De Danske Spritfabrikker) was suspended in
10 mL of TSB and the suspension was kept for 60 minutes at
37~C and a sample was collected for determination of the
viable yeast cell count (CFU per g) which was found to be
about 1.8 x 109 CFU of yeast per mL. A loaf of the bread was
injected essentially as described in Example 2 but using 1 mL
of yeast suspension and kept at room temperature for about 24
hours.
After keeping, a slice of about 1.5 cm was cut and 10 g
hereof around the site of injection was collected for the
determination of the viable yeast count according to the
following method: serial dilutions were prepared as described
~094/00019 ~1 3 8 7 6 g PCT/DK93/00207
2S
in Example 2 and appropriate dilution plated onto Malt agar
(Oxoid CM591) and the plates were incubated aerobically for 3
days at 25C followed by counting of the yeast colonies.
-- The viable count of yeast in the slice of ryebread as exami-
ned was 1.4 x 1o8 per g.
