Note: Descriptions are shown in the official language in which they were submitted.
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BACTERIAL STRAIN, PROCESSED PLANT EXTRACTS,
COMPOSITIONS CONTAINING SAME, PROCESSES FOR THEIR
PREPARATION AND THEIR THERAPEUTIC AND INDUSTRIAL
APPLICATIONS
FIELD AND BACKGROUND OF THE INVENTION
The present invention relates to (i) a non-pathogenic probiotic
microorganism and its probiotic/therapeutic uses; (ii) a formulation
comprising
an aqueous solution of a volatile fraction (VF) prepared from the extract of
at
least one plant derived material and its therapeutic uses; (iii) a process of
manufacturing the formulation from the plant derived material; (iv) a
probiotic
composition comprising the non-pathogenic probiotic microorganism of the
invention and/or other ~probiotic microorganisms) and the formulation of the
invention, and its probiotic/therapeutic uses; (v) a composition for
industrial
applications comprising the formulation of the invention and microorganisms)
of industrial applicability; and (vi) industrial processes and apparatuses in
which the latter composition is used.
Under normal conditions, the gastrointestinal (GI) tract microflora
contributes significantly to the health and well being of an individual. It is
well known that the microflora is a complex and diverse population of
microorganisms, which may have both beneficial and harmful effects on the
individual. Under normal gestation conditions, the fetus ih ute~o is sterile,
but
on passage through the vagina during birth it acquires microorganisms
resulting in the formation of a gut microflora. The final indigenous gut
microflora which stabilizes in the gut is a very complex collection of over
one
thousand different types of microorganisms, consisting of about 400 different
types of bacteria [Fuller R. J. Applied Bacteriology 66:365-378 (1989)]. The
composition of the gut microflora is determined by both host and microbial
factors, and although there are a lot of bacteria which can survive and grow
in
the GI tract, there are many which cannot. In addition, the surviving
organisms have to avoid the effect of peristalsis which normally flushes out
the
bacteria with the food. This may be achieved by the bacteria immobilizing
themselves by attachment to the gut wall, and/or by multiplying at a rate that
exceeds the rate of removal by peristalsis.
In general, the presence of gut microflora is symbiotic as the microflora
not only assists in disintegrating some undigestable foods, it also protects
the
individual~from infections caused by pathogens. This latter phenomenon has
been described under such names as 'bacterial antagonism', 'bacterial
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interference', 'barrier effect', 'colonization resistance', 'competitive
exclusion' and many others.
The gut protective microflora is very stable. However, it is less
effective in the young, elderly and the compromised patient. Further, it can
be
influenced by certain dietary and environmental factors, the three most
important being excessive hygiene, antibiotic therapy and stress.
Under conditions where the balance of the gut microflora is adversely
affected, probiotics become of potential value in restoring the gut microflora
and enabling the individual host to return to normal.
Probiotics are a class of microorganisms defined as live microbial
organisms that beneficially affect the animal and human hosts. The beneficial
effects include improvement of the microbial balance of the intestinal
microflora or improving the properties of the indigenous microflora. The
beneficial effects of probiotics may be mediated by a direct antagonistic
effect
against specific groups of organisms, resulting in a decrease in numbers, by
an
effect on their metabolism or by stimulation of immunity. Probiotics may
suppress viable counts of an undesired organism by producing antibacterial
compounds, by competing for nutrients or for adhesion sites. Further, they
may alter microbial metabolism by increasing or decreasing enzyme activity or
they may stimulate the immune system by increasing antibody levels or
increasing macrophage activity.
W095/16461 describes a probiotic composition of anaerobic bacteria
effective in controlling or inhibiting Salmonella colonization in domesticated
animals. The probiotic composition includes populations or cultures of 29
substantially biologically pure bacteria, iate~ alia, E. coli. However, the
suppression of the pathogen by the probiotic composition described in this
PCT publication requires the combined action of a large number of bacterial
strains.
W097/35596 describes the administration of a freshly prepared
probiotic mixture obtained by mixing a powder containing Lactobacillus
reute~i, Lactobacillus acidophilus and Bifidobacte~~ium i~faatis with a
liquid.
The mixture is described to be effective in preventing infectious diarrhea or
diarrhea caused by antibiotic therapy in humans. The freeze-dried live
bacteria
are, however, in anabiotic state. The need to wet the microorganism before
administration, in order to reinstate its vitality, is a disadvantage, since
normally many bacteria do not survive the re-hydration. Moreover, the.
surviving organisms are not immediately metabolically active, and cannot
survive the extreme, acidic conditions of the stomach. Furthermore, when
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administered to a recipient with diarrhea, the rate of their removal from the
gut
may exceed the rate of reinstation of viability, resulting in minimal or no
beneficiary effect.
Preservation of viability and conservation of the activity of probiotic
organisms by their formulation is the issue of numerous publications.
WO98/26787 describes the enhancement of a resident population of lactic
acid-producing microorganisms, preferably lactobaccillii, in the GI tract of
an
animal by providing the same with ~i-glucan, optionally in combination with
prebiotic and/or probiotic microorganisms.
W097/34591 also describes the enhancement of resident population of
microorganisms, or the suppression of the undesired resident population at a
selected site of the GI tract of an individual, by providing the individual
with a
selected modified or unmodified starch or mixtures thereof, which act as
carrier for one or more probiotic microorganisms and as a growth or
maintenance medium for the microorganisms. The probiotic elements are
bound to the caxrier in a manner so as to protect the microorganisms during
passage to the laxge bowel or other regions of the GI tract.
Microorganisms are used in a variety of industrial processes. These
processes can be divided into continuous processes and batch processes. In
continuous processes, such as a continuous aerobic or anaerobic fermentation
line, used for the production of a variety of fermentation products,
including,
for example, ethanol and methanol, a fermentable raw material is continuously
fed into the line and the fermentive product is continuously collected from
the
line, in a controlled fashion, such that the microorganism population in the
line
is sustained throughout the process. In batch processes, such as, but not
limited to, batch aerobic or anaerobic fermentation, biodegradation of oil in,
for example, oil spills, and the like, a fermentable raw material is mixed
with a
population of microorganisms) so as to instate fermentation. Both in
continuous fermentation and in batch fermentation processes, it is desired, in
~ some cases, to start or reinstate the process with a large and viable
population
of the relevant microorganism(s). For example, when biodegrading oil, it is
advantageous to start the process with an as large as possible population of
the
microorganisms Pseudomonas spp. or Alcaligenes spp., for example, so as to
minimize the time for complete biodegradation of the oil. When using filters
enriched with microorganisms) for biodegradation of organic fumes or
volatiles, the result is a continuous process in which polluting compounds are
degraded into harmless compounds. However, the microorganisms present in
the filter depend for their survival on continuous supply of organic fumes or
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valatiles. Nevertheless, in some instances the supply of organic fumes or
valatiles is discontinued and, as a result, the microorganisms) are lost.
Under
such circumstances, reinstation of an effective viable amount of
microorganisms) in the filter is required in a short period of time, so as
permit
immediate restoration of a functional filter.
There is thus a widely recognized need for, and it would be highly
advantageous to have, a formulation in which microorganisms, including
probiatic microorganisms and microorganisms useful in a variety of industrial
applications, could be maintained viable and in a metabolically active form
for
long periods of time.
While reducing the present invention to practice, it has been
unexpectedly found that a single species of a non-pathogenic probiotic
microorganism derived from E. coli is, alone, capable of restoring normal GI
flora of man and of a variety of mammals and avians. It has else been
surprisingly found that this microorganism, as well as other microorganisms,
could be preserved for long periods of time, in a viable and metabolically
active form, in a formulation comprising water solution of volatile fractions)
of various plant extracts. A probiotic composition comprising the probiotic
organism suspended in the formulation was found to be effective in the
treatment and prevention of various gastrointestinal disorders. It has further
been unexpectedly found that the formulation per se is effective as a body
weight gain enhancer and as an immuno-stimulator in mammals and avians.
SUMMARY OF THE INVENTION
According to one aspect of the present invention there is provided an
Esche~ichia coli strain BU-230-98 ATCC Deposit No. 20226 (DSM 12799).
According to another aspect of the present invention there is provided a
probiotic composition comprising viable Esche~ichia coli strain BU-230-98
ATCC Deposit No. 20226 (DSM 12799) and a formulation for maintaining
viability of the Esche~ichia coli strain. The probiotic composition preferably
further comprising at least one flavouring agent. Preferably, the formulation
includes at least one volatile fraction (VF) of a plant extract prepared by
steam
distillation of a plant (e.g., beet, dill, parsley or grapefruit) extract
under
reduced pressure and at a bath temperature preferably not exceeding 38
°C.
The probiotic composition of the present invention may be identified
for preventing or treating gastro-enteric infections or disorders, maintaining
or
reinstating normal gastro-intestinal microflora, preventing or treating
diarrhea,
preventing or treating gastro-enteric infection caused by an enteric pathogen,
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such as a Gram negative bacterium or Gram positive bacterium, preventing or
treating gastro-enteric Salmonella infection, preventing or treating
infectious
diarrhea, caused by, fox example C. difficile, Salmonella, particularly S.
Shigella, Campylobacter, E. coli, Py~oteus, Pseudomo~cas or Clostridium,
S chronic diarrhea or diarrhea resulting from antibiotic therapy, radiotherapy
or
chemotherapy, and/or for normalizing the physiological activity of the
gastrointestinal tract.
According to yet another aspect of the present invention there is
provided a formulation comprising at least one volatile fraction (VF) of a
plant
extract, the volatile fraction is prepared by steam distillation of the plant
extract under reduced pressure and at a bath temperature not exceeding 38
°C.
The plant extract is preferably obtainable from a plant organ selected from
the
group consisting of leaves, stems, roots and fruit. The plant can be a
vegetable, such as soy bean, alfalfa, garlic, beet and cabbage, or a herb,
such as
parsley, mint and dill. The formulation may further include a beehive product,
such as, but not limited to, propolis. The formulation can serve as a
veterinary
feed or food additive for enhancing animal weight gain in a recipient. It can
also serve to preserve viable bacteria as is further described herein.
Hence, according to an additional aspect of the present invention there
is provided a process of preparing a volatile fraction of a plant, comprising
the
steps of (a) grinding a plant derived material to give a plant biomass; (b)
mixing the plant biomass with water and stirring at ambient temperature; (c)
steam distilling the mixture obtained in step (b) under reduced pressure and
at
a bath temperature not exceeding 38°C; and (d) collecting volatile
fraction
obtained from the steam distillation. Preferably, the reduced pressure is of
5-10 mbar. This formulation can be used as a food additive, a feed additive
and has unexpected therapeutic uses, as well as advantageous bacteria
preservation properties. A therapeutic formulation containing the volatile
fraction of a plant is also disclosed and may be prepared by mixing at least
one
volatile fraction (VF) of a plant extract with physiologically or veterinary
acceptable additives, carriers or diluents. Preferably, the volatile fraction
is
prepared by steam distillation of the plant extract under reduced pressure and
at a bath temperature not exceeding 38 °C.
Thus, according to still another aspect of the present invention there is
provided a probiotic composition comprising (a) an effective amount of at
least one viable probiotic microorganism having a beneficial biological or
therapeutic activity in the gastrointestinal tract; and (b) at least one
volatile
fraction (VF) of a plant extract preferably prepared as described herein. The
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probiotic microorganism can be E. coli, preferably, strain BU-230-98, ATCC
Deposit No. 202226 and it can be identified for treatment or prevention of any
of the above disorders/pathologies/diseases/syndromes and in addition or as an
alternative in can be identified for treatment or prevention of dyspeptic
symptoms, for enhancing the immune response in a patient suffering from an
immune disorder, resulting from, for example, immune-response suppression
therapy.
According to still another aspect of the present invention there is
provided a composition comprising (a) at least one microorganism, such as,
Esche~ichia spp., Alcaligehes spp., A~throbacte~ spp., Bifidobacte~ium spp.,
Lactobacillus spp., Lactococcus spp., Nit~osomonas spp. and Pseudomonas
spp.; and (b) at least one volatile fraction (VF) of a plant extract, the at
least
one volatile fraction is selected so as to sustain viability of the at least
one
microorganism for at least 2 months, preferably at least 3-6 months, more
preferably 6-12 months or more at room temperature.
According to another aspect of the present invention there is provided a
process of growing Esche~ichia coli strain BU-230-98 ATCC Deposit No.
20226 (DSM 12799), the process comprising the step of seeding a starter
cloture of Esche~ichia coli strain BU-230-98 ATCC Deposit No. 20226 (DSM
12799) into a growth media and growing the EschericlZia coli strain up to
optical density of 15-30 at 650 nm.
According to yet another aspect of the present invention there is
provided a process of manufacturing a probiotic composition, the process
comprising the step of suspending viable Esche~ichia coli strain BU-230-98
ATCC Deposit No. 20226 (DSM 12799) in a formulation for maintaining
viability of the Esche~ichia coli strain. Preferably, the formulation includes
at
least one volatile fraction (VF) of a plant extract, prepared, preferably by
steam distillation of the plant extract under reduced pressure and at a bath
temperature not exceeding 38 °C, obtained from, for example, a plant
organ
selected from the group consisting of leaves, stems, roots and fruit, either a
vegetable and a herb, such as soy bean, parsley, mint, dill, alfalfa, garlic,
beet
or cabbage.
According to still another aspect of the present invention there is
provided a process of preparing a probiotic composition, the process
comprising the step of suspending an effective amount of at least one viable
probiotic microorganism having a beneficial biological or therapeutic activity
in the gastrointestinal tract in an formulation containing at least one
volatile
fraction (VF) of a plant extract.
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According to an additional aspect of the present invention there is
provided a process of preparing a composition including viable
microorganisms, the process comprising the step of suspending at least one
microorganism in a formulation containing at least one volatile fraction (VF)
of a plant extract, the at least one volatile fraction is selected so as to
sustain
viability of the at least one microorganism for at least 2 months at room
temperature. The microorganism can be, for example, Esche~ichia spp.,
Alcaligehes spp., A~th~obacter~ spp., Bifidobacterium spp., Lactobacillus
spp.,
Lactococcus spp., lVit~osomonas spp. or Pseudomouas spp.
According to yet an additional aspect of the present invention there is
provided a dispenser of microorganisms comprising a reservoir and a
dispensing mechanism being connected thereto, the reservoir housing at least
one microorganism in a formulation containing at least one volatile fraction
(VF) of a plant extract, the at least one volatile fraction is selected so as
to
sustain viability of the at least one microorganism for at least 2 months at
room
temperature.
According to still an additional aspect of the present invention there is
provided a method of biocontrol of an organism, the method comprising the
step of spreading in an endemic area at least one viable microorganism capable
of biocontrolling the organism, the at least one viable microorganism being in
a formulation containing at least one volatile fraction (VF) of a plant
extract,
the at least one volatile fraction is selected so as to sustain viability of
the at
least one microorganism for at least 2 months at room temperature.
According to a further aspect of the present invention there is provided
a method of eliminating an oil pollution, the method comprising the step of
spreading in a polluted area at least one viable microorganism capable of
degrading oil, the at least one viable microorganism being in a formulation
containing at least one volatile fraction (VF) of a plant extract, the at
least one
volatile fraction is selected so as to sustain viability of the at least one
microorganism for at least 2 months at room temperature.
According to yet a further aspect of the present invention there is
provided a method of restoring organic fumes degrading microorganism
population in a biofilter, the method comprising the step of dispensing onto
the
filter at least one viable microorganism capable of degrading organic fumes,
the at least one viable microorganism being in a formulation containing at
least
one volatile fraction (VF) of a plant extract, the at least one volatile
fraction is
selected so as to sustain viability of the at least one microorganism for at
least
2 months at room temperature.
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According to still a further aspect of the present invention there is
provided a method of preparing a starter for a fermentation process, the
method comprising the step of growing a sufficient amount of a starter
microorganism and suspending the starter microorganism in a formulation
containing at least one volatile fraction (VF) of a plant extract, the at
least one
volatile fraction is selected so ~as to sustain viability of the starter
microorganism for at least 2 months at room temperature.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention is herein described, by way of example only, with
reference to the accompanying drawings. With specific reference now to the
drawings in detail, it is stressed that the particulars shown are by way of
example and for purposes of illustrative discussion of the preferred
embodiments of the present invention only, and are presented in the cause of
providing what is believed to be the most useful and readily understood
description of the principles and conceptual aspects of the invention. In this
regard, no attempt is made to show structural details of the invention in more
detail than is necessary for a fundamental understanding of the invention, the
description taken with the drawings making apparent to those skilled in the
art
how the several forms of the invention may be embodied in practice.
In the drawings:
FIG. 1 is a schematic depiction of a dispenser according to the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is of (i) a non-pathogenic probiotic
microorganism and its probiotic/therapeutic uses; (ii) a formulation
comprising
an aqueous solution of a volatile fraction (VF) prepared from the extract of
at
least one plant derived material and its therapeutic uses; (iii) a process of
manufacturing the formulation from the plant derived material; (iv) a
probiotic
composition comprising the non-pathogenic probiotic microorganism of the
invention and/or other probiotic microorganisms) and the formulation of the
invention, and its probiotic/therapeutic uses; (v) a composition for
industrial
applications comprising the formulation of the invention and microorganisms)
of industrial applicability; and (vi) industrial processes and apparatuses in
which the latter composition is used.
The principles and operation of the present invention may be better
understood with reference to the drawings and accompanying descriptions.
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Before explaining at least one embodiment of the invention in detail, it
is to be understood that the invention is not limited in its application to
the
details set forth in the following description or exemplified by the Examples.
The invention is capable of other embodiments or of being practiced or carried
out in various ways. Also, it is to be understood that the phraseology and
terminology employed herein is for the purpose of description and should not
be regarded as limiting.
In one aspect, the present invention relates to a probiotic microorganism
being a non-pathogenic bacterium derived from E. coli, having a beneficial
physiological and/or therapeutic activity in, for example, the GI tract and
deposited at the ATCC under deposit No. 202226 and at DSMZ under deposit
No. 12799.
"Probiotic" is used herein as an adjective to describe an isolated
bacteria having the property of inhibiting the growth of at least one
pathogen.
The test of an inhibition used herein was an i~ vitro test on solid medium in
which culture supernatants of candidate isolated bacteria were observed for
their property of inhibiting the growth of a pathogen when applied to the
surface of the solid medium. Typically, a paper disc impregnated with the
culture supernatant of a candidate probiotic strain was placed on the surface
of
an agar plate seeded with the pathogen. Probiotic bacterial supernatants
caused a ring of clear agar or of reduced growth density indicating inhibition
of the pathogen in the vicinity of the disc. There are other tests for
inhibition
which are available or could be devised, including direct growth competition
tests, ih viti~o or in vivo which can generate a panel of probiotic bacteria
similar
to that described herein. The bacterial strains identified by any such test
are
within the category of probiotic bacteria, as the term is used herein.
The probiotic bacteria of the present invention may serve as a food or
feed additive, so as to provide food or feed product supplemented with viable
Esche~ichia eoli strain BLT-230-98 ATCC Deposit No. 20226 (DSM 12799).
A food product of the present invention may further include at least one milk
derived substance and may be selected from the group consisting of a cheese
and a yogurt.
The present invention further relates to a formulation comprising at
least one volatile fraction (VF) of a plant extract, the volatile fraction
being
prepared by distillation of the plant extract under reduced pressure and at a
bath temperature typically not exceeding 38 °C.
The plant matter from which the volatile fraction may be obtained may
be any suitable plant part such as fruit, leaf, stem or root. Many plants are
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suitable as a source for the volatile fractions, for example apple, citrus,
soy
bean, beet, cabbage, garlic and alfalfa, as well as herbs such as parsley,
mint
and dill. The formulations of the invention may optionally further comprise a
suitable amount of a volatile fraction of an apicultural product such as
honey,
5 propolis or other beehive product, which may be prepared in the same manner
as described herein for obtaining volatile fractions from plant extracts.
As will be described in more detail in the following Examples, the
formulation itself may be used as a food/feed additive. It has been found by
the inventors to have a weight gain enhancing activity and it constitutes an
10 aspect of the invention. According to a particular embodiment of this
aspect
of the invention, the animal weight gain enhancing formulation or feed
additive comprises distilled water and volatile fractions of alfalfa, soy
beans,
beet and dill, preferably at a volume ratio of the volatile fractions of
2:8:.1:4.
Alternatively, a formulation or feed additive of the present invention
comprises distilled water and volatile fractions of carrot, beet, dill and
grapefruit (1 part each) per 100 parts of water. Still alternatively, a
formulation or food additive of the present invention comprises distilled
water
and volatile fractions of beet, dill and parsley (1 part each) per 100 parts
of
water.
In a further aspect, the invention relates to a composition comprising
the above formulation of the invention and at least one viable microorganism,
either a probiotic organism having a therapeutic or beneficial biological
activity in, for example, the GI tract of human and/or animal, to thereby
provide a probiotic composition, or an organism having industrial
applications,
to thereby provide a composition of industrial applicability.
A particular advantage of the probiotic and industrial compositions of
the invention stems from the fact that they are liquid preparations. Being
under biologically active conditions, the formulation serves also as a
supportive medium for living bacteria, as opposed to lyophilized formulations
where the bacteria are in an anabiotic condition. As a result, the probiotic
composition of the invention, for example, is active immediately following
oral administration, beginning with the upper portion of the GI tract, where
primary effects of the majority of intestinal pathogens take place, causing
development of adverse gastro-enteric syndromes. The probiotic compositions
of the invention may also be used as body weight-increasing preparations or
food/feed additives. Similarly, the industrial composition of the invention
can
be used to efficiently reinstate a population of microorganisms of industrial
applicability.
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Thus, it is noted that, for probiotic applications, probiotic bacteria other
than such belonging to the strain BU-230-98 (ATCC Deposit No. 202226, also
deposited at the DSM under No. 12799), may be used with the plant material
derived formulation of the invention. Such probiotic bacteria have a very
broad spectrum of antagonistic activity. They also belong to the same
phylogenetic group of the majority of intestinal pathogens and share the same
systems of survival. Therefore, the suppression and exclusion of intestinal
pathogens may include many different mechanisms, for example, secretion of
antagonistic material, competition for utilization of nutrients and
competition
for adhesion receptors. Thus, any non-pathogenic bacteria which comply with
these criteria, may be used in the probiotic compositions of the invention.
According to a further aspect of the invention, the probiotic
compositions of the invention may be used for preventing or treating
gastro-enteric infections. Term 'gastro-enteric infection' is to be taken to
mean any infection caused an enteric pathogen, including, ihte~ alia, Gram
negative and Gram positive bacteria. By improving the general balance and
health of the GI tract, the formulations and probiotic compositions of the
invention may be instrumental in prophylaxis of also GI infections caused by
yeast, viruses and protozoa.
The term 'therapeutically effective amount' or 'effective amount' for
purposes herein is the amount determined by such considerations as are known
in the art. The amount must be sufficient to enable the efficient restoration
of
the GI microflora thus leading to the normalization of the function of the GI
tract.
In a particular embodiment, the probiotic composition for preventing
the development of gastro-enteric infections comprises the formulation of the
invention made from distilled water and volatile fractions of alfalfa, soy
beans,
beet, dill and mint as defined herein, at a volume ratio of, e.g., 5:1:5:15:1,
and
a therapeutically effective amount of a probiotic bacteria such as E. coli
ATCC Deposit No. 202226 (identical with DSM 12799) or any other probiotic
bacteria.
A specific example for an gastro-enteric infection is that caused by
Salmonella and the invention is of particular advantage in preventing or
treating gastro-enteric infections caused thereby.
Further, the food additive or formulation of the invention, alone or in
combination with an effective amount of a probiotic microorganism, such as
the E. coli strain ATCC Deposit No. 202226 (identical with DSM I2799),
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may be used for treating or preventing infectious diarrhea, chronic diarrhea
or
diarrhea caused by antibiotic or chemo-therapy.
According to a further specific embodiment of the invention, such a
probiotic composition for treating diarrhea may comprise distilled water and
volatile fractions of alfalfa, soy beans, beet, dill and mint at a volume
ratio of,
e.g., 5:1:5:15:1, and a therapeutically effective amount of the ATCC No.
202226 (DSMZ 12799).
The infectious diarrhea may be caused by numerous factors, for
example, by a microorganism selected from C. di~cile, Salnaohella,
particularly S. Shigella, Campylobacte~, E. coli, Proteus, Pseudomohas,
Clostridium, enteric Staphylococcus. These are but few of many infecting
agents.
Y'et further, the probiotic compositions of the invention may be used for
effectively restoring the GI microflora in a subject in need of such treatment
which leads to the normalization of the function of the GI tract. Such
compositions may include, for example, distilled water and volatile fractions
of alfalfa, soy bean, beet, dill, mint, parsley and cabbage, preferably at a
ratio
of volatile fractions of, e.g., 1:5:5:2:2:1, and a therapeutically effective
amount
of the probiotic bacteria ATCC Deposit No. 202226 (DSM 12799).
Other purposes for which the probiotic compositions of the invention,
comprising at least one probiotic bacteria having a therapeutic effect in, for
example, the GI tract, include alleviation of lactose intolerance in subjects
suffering from lactose intolerance, treatment of enterocolitis, treatment of
constipation, for reduction of cholesterol levels in the blood, for treatment
of
dyspeptic symptoms, and/or for stimulation of the immune system in subj ects
suffering from an immune system disorder, which may be an immune disorder
caused by immuno-suppressive therapy.
In a different aspect, the invention relates to a process for preparing a
volatile fraction of a plant extract, which process comprises the steps of
(a) grinding plant derived matter to obtain a plant biomass; (b) mixing the
plant biomass obtained in step (a) with water at a weight proportion of
preferably 3 parts water to 1 part of the plant biomass and stirring the same
for
at least 2 hours at ambient temperature; (c) distilling the mixture obtained
in
step (b) under reduced pressure and at a bath temperature preferably not
exceeding 38 °C; and (d) collecting the volatile fraction obtained from
the
steam distillation, which fraction may further be diluted in a suitable
buffer.
The volatile fraction of the invention may be mixed with water to give
the food/feed additive or formulation of the invention. The food/feed additive
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or formulation of the invention may also be prepared by mixing more than one
plant volatile fraction obtained by the process of the invention. This mixture
may be further mixed with water.
The volatile fractions may be prepared from may any suitable fruit,
vegetable, leaf, stem or root of a plant. The plant can be, for example,
apple,
citrus fruit, soy bean, beet, garlic, cabbage or alfalfa, or a herb such as
parsley,
mint or dill. It should be noted that when appropriate, the formulation may
further comprise volatile fractions from apicultural products such as honey or
propolis or other beehive products. These volatile fractions may be prepared
in the same manner of the plant extract volatile fractions.
According to the process of the invention, the distillation step is
preferably carried out under reduced pressure of 5-10 mbar.
As indicated hereinbefore, the food/feed additive or formulation of the
invention may be further combined with at least one probiotic or industrial
agent, to give the probiotic or industrial compositions of the invention.
Thus,
the process of preparation of the invention may also further comprise the step
of suspending at least one viable probiotic or industrial microorganism in the
volatile fraction obtained in said step (d) or in a mixture of such volatile
fractions obtained as described above.
According to a particular process of the invention, the probiotic
microorganism suspended may be the novel bacterium of the invention,
derived from E. coli and deposited at the ATCC under Deposit No. 202226
(and at DSM under deposit No. 12799).
Finally, the invention relates to the use of a volatile fraction (VF) of a
plant extract in the preparation of a food/feed additive, wherein the VF is
prepared by steam distillation of said plant extract or from apiculture
product
extract such as honey or propolis at a bath temperature not exceeding 3~
°C.
An additional aspects of the invention include a process of preparing a
composition including viable microorganisms. The process is effected by
suspending at least one microorganism in a formulation containing at least one
volatile fraction (VF) of a plant extract. The formulation is selected so as
to
sustain viability of the microorganisms) for at least 2 months at room
temperature. The microorganism of choice may be probiotic or have industrial
applicability. Organisms which can be maintained in a formulation of the
present invention include Escherichia spp., Alcaligehes spp., Arthrobacter
spp., Bifidobacte~ium spp., Lactobacillus spp., Lactococcus spp.,
Nit~osomonas spp. and Pseudomonas spp.
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14
As shown in Figure l, in another aspect the present invention provides a
dispenser 10 of microorganisms. Dispenser 10 includes a reservoir 12 and a
dispensing mechanism 14 connected thereto. Reservoir 12 houses at least one
microorganism in a formulation containing at least one volatile fraction (VF)
of a plant extract. The formulation is selected so as to sustain viability of
the
microorganisms) for at least 2 months at room temperature. Reservoir 12 may
be refrigerated, say to about 2-20 °C, preferably to about 4 °C.
Dispenser 10
may be an airborne dispenser, so as to allow dispersion of pest control
bioagents. The dispenser may be hand held. In any case, the dispenser may be
designed to automatically dispense a predefined volume therefrom, e.g.,
periodically. To this end, dispenser 10 may include a programmable or preset
control mechanism 16, valves 18, etc.
Thus, according to another aspect, the present invention provides a
method of biocontrol of an organism which is effected by spreading in an
endemic area at least one viable microorganism capable of biocontrolling the
organism. The viable microorganisms) are present in a formulation
containing at least one volatile fraction (VF) of a plant extract selected so
as to
sustain viability of said at least one microorganisms) for at least 2 months
at
room temperature.
Still according to another aspect the present invention provides a
method of eliminating an oil pollution which is effected by spreading in a
polluted area at least one viable microorganism capable of degrading oil, such
as Pseudomonas spp. The viable microorganisms) is present in a formulation
containing at least one volatile fraction (VF) of a plant extract selected so
as to
sustain viability of said at least one microorganism for at least 2 months at
room temperature.
Yet according to another aspect the present invention provides a method
of restoring organic fumes degrading microorganism population in a biofilter
by dispensing onto the filter at least one viable microorganism, such as
Pseudomonas spp., Nitrosomohas spp., or A~th~~obacte~ spp. capable of
degrading organic fumes. The viable microorganisms) is present in a
formulation containing at least one volatile fraction (VF) of a plant extract
selected so as to sustain viability of said at least one microorganism for at
least
2 months at room temperature.
Still according to another aspect of the present invention there is
provided a method of preparing a starter for a fermentation process by growing
a sufficient amount of a starter microorganism, such as, Lactococcus spp.,
Escherichia spp., Bifidobacte~ium spp., Lactobacillus, Lactococcus spp. or
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Pseudomohas spp. and suspending the starter microorganism in a formulation
containing at least one volatile fraction (VF) of a plant extract selected so
as to
sustain viability of said starter microorganism for at least 2 months at room
temperature.
5
Additional objects, advantages, and novel features of the present
invention will become apparent to one ordinarily skilled in the art upon
examination of the following examples, which are not intended to be limiting.
Additionally, each of the various embodiments and aspects of the present
10 invention as delineated hereinabove and as claimed in the claims section
below
finds experimental support in the following examples.
EXAMPLES
Reference is now made to the following examples, which together with
15 the above descriptions, illustrate the invention in a non limiting fashion.
Example 1
Preparation of a "volatile fraction" of plant extracts
or extracts from apiculture products
Fresh vegetables obtained from commercial markets were thoroughly
washed by tap water, chopped and finely ground in an industrial blender.
Distilled water was added to the vegetable biomass at the proportion of 3
parts
water to 1 part of the vegetable mass by weight and left under stirring for at
least 2 hours at ambient temperature.
The mixture was then transferred into the evaporating flask of a rotatory
evaporator and was evaporated at a reduced pressure (5-10 mbar) under such
conditions that the temperature of the water bath did not exceed 3 ~
°C, and
that of the condenser inlet was 2-5 °C. About 1 liter of the condensate
(volatile fraction) were collected per 2.5 kg of plant biomass. This material
could be preserved under refrigeration for at least 12 months without loosing
its properties, as is further exemplified hereinunder.
The same procedure may be carried out, replacing the plant material
with apiculture products such as honey or propolis.
Usually, each "volatile fraction" was prepared from a single kind of
vegetable and used in different dilution to prepare different mixtures for
vanous purposes.
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Example 2
Isolation and growth of the probiotic organism
E. coli ATCC Deposit No. 202226
The probiotic organism E. coli (deposited at the ATCC under No.
202226 on May 3, 1999 and at the DSM under No. 12799 on May 4, 1999)
was isolated from E. coli M-17 by sequential transfer of isolates initiating
from
long term (2 months) survivors preserved in a formulation containing volatile
fractions of plant derived material as is described under Example 1 above at
37
°C and selecting for isolates capable of competition with pathogens as
is
further detailed below. The probiotic organism E. coli (deposited at the ATCC
under No. 202226 on May 3, 1999 and at the DSM under No. 12799 on May 4,
1999) was grown in the following medium: (NH4)2504 - 5 g/liter, I~H2P04 -
13 g/liter, Na2HP04 - 13 g/liter, MgCl2 - 3 g/liter, CaCl2 - 0.3 g/liter,
yeast
extract - 10 g/liter, Soy peptone - 10 g/liter and glucose - 5 g/liter.
Additional
nutrients (yeast extract - 1 g/liter, Soy peptone - 2.5 g/liter and glucose -
90
g/liter) were continuously added following the growth of the culture in such a
way that the glucose concentration in the fermentation broth was kept at a
level
of 2 g/liter. The pH of the fermentation broth was kept neutral by the
continuous addition of 4N NH40H. Culturing was carried out at 30 °C in
a
standard fermentation vessel with aeration of 0.5 wm for 16 hours, when the
growth became confluent. This procedure resulted in 1010-1011 cells/ml. The
E. coli cells were harvested by centrifugation, resuspended in saline and
re-precipitated. The microbial biomass could be kept in saline in the
refrigerator for 48 hours without loosing viability.
Example 3
Preparation of foodlfeed additive formulation for the
accelerated increase of body weight in mammals and avians
The mixture contained volatile fractions of alfalfa - 50 ml/liter, soy
beans - 200 ml/liter, beet - 25 ml/liter and dill - 100 ml/liter, prepared as
described under Example 1 above. The balance was made up by the distilled
water.
Example 4
Preparation of a food supplement for the normalization
of the function of the human GI tract
The mixture contained volatile fractions of: alfalfa - 50 ml/liter, soy
beans - 10 ml/liter, beet - 50 ml/liter, dill - 50 ml/liter, mint - 20
ml/liter,
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t> 17
parsley - 20 ml/liter and cabbage - 10 ml/liter, prepared as described under
Example 1 above. The balance was made up by distilled water. E. coli
(ATCC 202226) cells (Example 2) were suspended in the mixture at a cell
concentration of 107 cells/ml. NaCI (e.g., 4-10 g/liter) may be optionally
added for the improvement of taste.
Example 5
Preparation of a feed additive formulation for
the proplzylaxis of GI infections in mammals and avians
The mixture contained volatile fractions of: alfalfa - 50 - ml/liter, soy
beans - 10 ml/liter, beet - 50 ml/liter, dill - 150 ml/liter and mint - 10
ml/liter,
prepared as described under Example 1 above. The balance was made by
distilled water. E. coli (ATCC 202226) cells (Example 2) were suspended in
the mixture at a cell concentration of 107 cells/ml. NaCI (4-10 g/liter) may
be
optionally added for the improvement of the taste.
Example 6
Antagonistic activity of E. coli (ATCC 202226) against
Sal~rzonella typhymirium (ATCC 14028)
Petri plates containing Modified Brilliant Green Agar, a selective
growth medium for Salmonella, were inoculated with S. typhimirium. A 9 mm
diameter well was made in the agar. A volume of the food supplement
(Example 4) was deposited in each well, and the plates were incubated for
24 hours at 35 °C. The same was repeated, but instead of the food
supplement,
the fluid obtained by its filtration through a microbiological filter membrane
(pore size of 0.45 Ean) was deposited in the well.
Around each well containing the feed additive an inhibition zone (10-17
mm) devoid of S. typhimirium colonies was observed. No inhibition zone was
observed around the wells containing the filtrate free of the probiotic
organism.
Example 7
Antagonistic activity of E. coli ATCC 202226 as well
as of its parent strain E. coli M 17 against Shigella sps.
Cultures of S. flex~eri, S. sohhei, E. coli (ATCC 202226) and M-17
were grown separately on a Nutrient Agar for 18-20 hours at 37 °C. All
the
cultures were harvested in saline and diluted to an optical density of 10
Klett
units. Aliquots of the diluted cultures of Shigella species (1 ml) alone or in
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combination with the diluted culture of E. coli (ATCC 202226) (1 ml) were
seeded in ventilation-cup test tubes containing Nutrient Broth (5 ml). The
tubes were incubated for 24 hours at 37 °C. 'The number of colony-
forming
units (CFU) of the pathogens and of E. coli (ATCC 202226) was determined
by plating the cultures on Nutrient agar. The CFU numbers of two Shigella
species in the pure culture and in mixed cultures with both probiotic E. coli
species are shown in Table 1 below.
Table 1
Probiotic Organism Growth of Shi ella
CFU/ml
S. exhe~i S. sonnel
E. coli ATCC 202226 <5x104 <5x104
E. coli M-17 1x106 2x106
Pure Shi ella culture 6x106 2x106
Example 8
~ipplication of the food supplement (Example 4) and of Colibacterin (dry
formulation of E. coli M 17) in hospitalized gastroenteritis patiefats
A group of 60 patients that developed gastroenteritis following
hospitalization was randomly divided into 3 sub-groups with a similar
distribution of sex, age and the severeness of gasroenteritis symptoms. All
patients received normal supportive treatment, including rehydration,
vitamins,
etc. The severity of condition did not require treatment with antibiotics. The
first group of 20 patients received 10 ml of the food supplement (Example 5) 3
times daily 30 minutes before the meals for 7 days. The second group of the
same number received Colibacterin (Colibacterinum siccum) as recommended
by the producer (one dose twice a day 30 minutes before the meal) and the
third group received no supplement at all.
The onset of normalization (in days) of the symptoms of gastroenteritis
in all groups were recorded and are shown in Table 2 below.
Table 2
S m toms Onset of the
normalization
of the s tom,
da s
Food su lementColibacterin Control
Bod tem erature 2.80.2 2.70.2 3.70.2
Intoxication 2.50.1 3.60.1 4.60.1
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Abdominal ain 3.30.2 5.10.2 6.10.2
Diarrhea 1.80.2 3.40.2 4.40.2
Table 2 (Continued)
Colibacterin (Colibacterinum siccum) is the freeze-dried preparation of
live E. coli M-17 produced by BIOMED Ltd., Moscow, Russia, and
recommended for use against diarrhea [Vidal Handbook: Pharmaceutical
preparations in Russia (N.B. Nikolaeva, B.P. Alperovich and V.N. Sovinov,
Eds.) AstraPharmService, 1997, Moscow, p. 275].
Example 9
Application of the food supplement (Example 4)
in patients with acute GI infections
Patients with severe GI infections of various etiologies: salmonellosis,
escherichiosis, shigellosis, staphylococcal infections and food intoxications
of
unknown etiology were included in the study. In all patients the
hospitalization was indicated by an acute onset of the disease and appearance
of acute gastroenteritis. The total of 186 patients were treated with the food
supplement and a similar group of 102 patients received standard treatment.
The onset of normalization (in days) of the symptoms of gastroenteritis
in all groups were recorded and are shown in Table 3 below.
Table 3
Symptoms Onset of normalization,
days
Stud ou Control
Fever 1.20.3 2.70.3
Weakness 1.60.2 2.90.3
Abdominal pain 1.50.2 2.40.3
Diarrhea 2.30.2 4.70.4
Da s in bed 4.80.3 6.70.4
In a separate study a group of 30 patients with intestinal typhoid was
treated with the food supplement. In 80 % of patients the symptoms of disease
disappeared within 3 days. Only in 3 cases the supplement treatment was
stopped due to the development of more severe chronic colitis.
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Example 10
Applications of the food supplement (Example 4)
in patients with GI disorders caused by antibiotics
Patients with severe GI disorders were divided in 2 groups. Group I
5 contained 48 patients with peptic ulcer disease who developed GI disorders
after antibiotic treatment against H. pylori. Group II contained 22 patients
that
developed GI disorders following antibiotic treatment of pneumonia.
The food supplement (5 ml) was given 3 times a day before meals for 7
days. In both groups symptoms of diarrhea disappeared in 2-3 days in all the
10 patients. After the administration of the supplement, complete
normalization
of intestinal microflora was observed in 84.5 % of the patients. It was
demonstrated by a dramatic increase in lactobacilli and Bifidobacteria,
reduction on the general count of E. ~ coli, complete disappearance of the
hemolytic E. coli and other pathogens such as Staphylococci, Proteus vulgaris
15 and even Cahdida sps. In the remaining 15.5 % of the patients, a
significant
improvement was observed.
Example 11
Application of the food supplement (Example S)
20 in patients with late radiation enterocolitis
The food supplement (10 ml, 3 times a day for 14 days, 30 minutes
before the meals) was given to 24 patients with womb, colon and gastric
cancer that developed enterocolitis following radiation therapy or a
combination of radiation and chemotherapy.
Prior to the use of the food supplement, all patients complained about
frequent and painful desire to defecate, liquid stool (4-12 times a day)
appearance of mucous (9 cases) and blood (3 cases) in the stool.
Two or three days after ingesting the supplement, all the patients noted
the lessening of pain and reduction in the number of defecations; the stool
had
a normal appearance. Four to five days later the diarrhea) syndrome was gone,
the appearance of blood and mucus ceased. The blood analysis showed a
strong improvement in blood indicators.
In the control group of a similar size the symptoms persisted.
Example 12
Application of the food supplement (Example 4) in AIDS patients
Patients suffering from AIDS frequently develop chronic diarrhea. A
group of such patients was given 10 ml of the food supplement (Example 5) 3
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21
times a day 30 minutes before the meals for 20 days. The control group
received no supplement. The results of the treatment are shown in Table 4
below.
Table 4
Parameter Food su lement Control
No. of patients 30 20
Average age, years 3 8~ 1 3 6~2
Daily defecation
frequency:
at the onset of the 3.40.3 3.60.3
Exp.
at the end of the 1.10.1 3.20.3
Exp.
one month after the 1.50.2 3.70.3
Exp.
Av. onset of normalization 6.00.7 remained abnormal
Patients receiving the food supplement showed normalization of
intestinal microflora: reduction in general number of coliforms, disappearance
of the hemolytic E. coli, increase in the numbers of Lactobacilli and
Bifidobacteria, reduction in Cahdida sps.
Example 13
Application of the foodlfeed additive (Example 3)
for accelerated weight increase in healthy piglets
Healthy piglets were administrated 3 ml per os of the food/feed additive
per piglet per day until weaning. The group receiving the feed additive gained
weight at weaning on the average 1.0 kg per piglet more than the control
group.
Example 14
Application of the feed additive (Example S) in healthy piglets
Several hundreds healthy piglets were administrated 3 ml per os of the
feed additive per piglet on the first and third day after delivery and at
weaning.
Mortality was cut down by 50 % compared with the control group receiving
standard prophylactic treatment with antibiotics. The group receiving the feed
additive gained weight at weaning on the average 0.39 kg per piglet more than
the control group. When the feed additive and antibiotic treatment were
compared in the same litter, the weight gain in the piglets obtaining the feed
additive was found higher by 2.4 kg than in the control.
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Example 1 S
Application of the feed additive (Example 4) iyz piglets showing diarrhea
Several hundred piglets showing diarrhea were given daily 5 ml per os
of the feed additive per piglet. The control group of the same size was
treated
with antibiotics: advocin, gentiamycin, amoxicillin. The symptoms of diarrhea
in the group receiving the feed additive disappeared within 1-2 days. No
mortality was observed, and piglets developed normally. Antibiotics stopped
diarrhea in the great majority of piglets but the piglets remained stunted in
their development.
About 70 retarded piglets, that received antibiotic treatment against
diarrhea for a week, and generally considered lost, were given the feed
additive for three days. All but two survived.
Example 16
Application of the feed additive (Example 4) in healtlzy calves
Day-old healthy calves were administrated 5 ml of the feed additive a
day in milk during 7 days. More than 95 % of the calves did not developed
diarrhea until they were 14 days old, when a few cases positive for Rotavirus
were diagnosed. Normally, the incidence of diarrhea in this farm is 20-30 %.
Example 17
Application of the feed additive (Example 6) in calves showing diarrhea
Calves developing diarrhea were given daily 10 ml of the feed additive
per animal with milk during 3-5 days. The symptoms of diarrhea disappeared
within 1-2 days in 90-95 % of calves. In the remaining 5-10 % the diarrhea
was caused by virus. These calves were treated with antibiotics with poor
results.
Example I8
Application of the feed additive (Example 6) in Izealtlzy lambs and goat kids
Day-old healthy lambs and goat kids were administrated 3 ml of the
feed additive a day in milk during 7 days. In some cases slight to moderate
signs of diarrhea were observed. These signs usually disappeared
spontaneously or were successfully treated with an increased dose of the feed
additive (5 ml). Normally the insidence of diarrhea in these farms is 50 % for
baby lambs and for goat kids.
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Example 19
Application of the feed additive (Example 5) in
lambs and goat kids slzowiug diarrhea
The feed additive was tested in a herd suffering from pathogenic E.
coli infections. In the year preceding the experiment, about 90 from 120 lambs
and goat kids died from diarrhea. Treatment with antibiotics was ineffective,
since the disease developed suddenly and with fast mortality. Lambs and goat
kids developing diarrhea were given daily 5 ml of the feed additive per animal
with milk during 3-5 days. A matching group of animals was treated, as
normally recommended, with antibiotics. The symptoms of diarrhea
disappeared within 1-2 days in about 90 % of the lambs and goat kids
receiving the feed additive. Their further development appeared normal. The
control group receiving antibiotics (gentamycin) showed much poorer results.
Diarrhea in this group persisted, calling for repeated treatment with
antibiotics.
The development of the control group was severely retarded.
Example 20
Application of the feed additive (Example S) in poultry
The feed additive was added to the drinking water with the average
uptake of 0.01 ml of the additive per day per chick during the breeding period
(42-49 days). An increase of 3.2 % in weight gain, accompanied by 4
improvement in food conversion was noted in controlled trials in broilers.
Excellent results were also obtained with turkeys of age 1 day to 6
weeks. Each bird received 0.01 ml of the additive per day. A weight gain of
over 10 % was observed, along with reduced mortality rate. Birds which still
exhibited diarrhea, were treated with 0.1 ml per day of the food additive,
without any treatment by antibiotics, and showed better recovery from the
control birds which were treated with only antibiotics.
Exafnple 21
Application of the feed additive (Example S) in dogs ayzd cats
Application of the feed additives in puppies resulted in cessation of the
symptoms of diarrhea within 24-48 hours.
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Example 22
Application of the feed additive (Example S) in mice;
Evidence for immunostimulation
Two-weeks-old C571B 1 mice were divided into 4 groups of 10
animals in each. Mice were deprived water from 4:00 pm to 8:00 am and then
given 15 ml of either water (groups 1 & 3) or the feed additive (groups 2 &
4).
This treatment was repeated for 2 weeks. At the end of this treatment mice
from groups 3 and 4 were subjected to laparotomy under ether narcosis. A cut
of 1.5 cm was made in the abdominal wall. Irritation of intestine was
carried out by a dry gause. All the mice were sacrificed 24 hours after
laparatomy; spleens were excised. Splenocytes were isolated and cultured on
RPMI medium .for 48-72 hours in the presence of phytohemagglutinin (PHA).
In the group treated with the feed additive the proliferative activity of
splenocytes reduced by laparotomy was recovered.
Splenocytes proliferation induced by PHA:
1. Control group/water 3060290
2. Control group/water/laparotomy 2120300
3. Test groupBioCocktail 2740370
4. Test groupBioCocktail/laparotomy 3040520
Example 23
Application of the food additive (Example 4) in human;
Evidence for imnzunostimulation
A number of ,.patients were operated for malignant tumors of womb,
cervix, stomach, and intestine. The gynecological patients (20 patients)
received follow-up radiotherapy; the abdominal patients (30 patients) received
chemotherapy. About half of the patients received the food additive twice
daily for the duration of therapy. In all patients receiving the food additive
there was observed a significant immunostimulation, as expressed in:
Gynecological tumors Abdominal tumors
Control Food Additive Control Food Additive
T-lymphocytes 0.03f0.01 O.l Of0.03 1.010.1 1.6f0.2
(CD3+, HLA-, DR+)
Lymphocyte blast
transformation
a.spontaneous 530f110 880f80 700f120 10701100
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b. PHA-induced 16001360 5720f2100 44001340 13370f3720
Phagocytes No. (%) 60.712.9 76.52.2 57.7f1.5 74.211.9
Example 24
Bacteria preservation effect of the formulation
E. coli cells were dispersed in a formulation prepared as described
5 under Example 1 above and was maintained for 6 months at room temperature.
Both at the beginning and at the end of the 6 months period,. samples derived
from the dispersion were seeded at appropriate consecutive dilutions on
nutrient broth agar plates. The number of colony formixlg units was
determined for both samples. The concentration determined was 1x107 in both
10 cases, showing the effect of the formulation in preserving microorganisms.
The above experiment was performed in parallel with Pseudomonas putida,
yielding the same results.
Although the invention has been described in conjunction with specific
15 embodiments thereof, it is evident that many alternatives, modifications
and
variations will be apparent to those skilled in the art. Accordingly, it is
intended to embrace all such alternatives, modifications and variations that
fall
within the spirit and broad scope of the appended claims. All publications,
patents and patent applications mentioned in this specification are herein
20 incorporated in their entirety by reference into the specification, to the
same
extent as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein by
reference.
In addition, citation or identification of any reference in this application
shall
not be construed as an admission that such reference is available as prior art
to
25 the present invention.
