U.S. patent application number 11/970060 was filed with the patent office on 2008-05-01 for use hydrolyzed medium containing microorganisms medicinally.
This patent application is currently assigned to TECHNOLOGY COMMERCIALIZATION CORP.. Invention is credited to Constantin V. Sobol, Yuzefa T. Sobol.
Application Number | 20080102061 11/970060 |
Document ID | / |
Family ID | 29734690 |
Filed Date | 2008-05-01 |
United States Patent
Application |
20080102061 |
Kind Code |
A1 |
Sobol; Constantin V. ; et
al. |
May 1, 2008 |
USE HYDROLYZED MEDIUM CONTAINING MICROORGANISMS MEDICINALLY
Abstract
A method for treating a vaginal disease by applying a hydrolyzed
product in a variety of ways including preferably intravaginal, as
well as oral, rectal, or transcutaneous administration, inhalation,
intravenous or intraperitoneal injection. The product is produced
by providing at least one solid plant product reduced to small
pieces and mixed with sugar and a biocompatible liquid for
fermentation at a temperature of between 15 and 55 degrees C. until
its acidity reaches the range of 300 to 900 Terner degrees.
Alternatively, the product is prepared by mixing in predetermined
amounts of sprouted grains, biocompatible liquid inoculated with at
least one from a variety of non-pathogenic microorganisms,
vegetables, fruits, berries, high protein products, herbs, sugar,
and a chemical element such as potassium. The mixture is then
fermented at a selected temperature for a specified length of time
to reach high acidity and high concentration of products of
bacterial metabolism. This invention relates to a hydrolyzed medium
used for the prevention of and treatment for urogenital infections,
cancer and endometriosis.
Inventors: |
Sobol; Constantin V.;
(St.-Petersburg, RU) ; Sobol; Yuzefa T.;
(St.-Petersburg, RU) |
Correspondence
Address: |
BORIS LESCHINSKY
P.O. BOX 72
WALDWICK
NJ
07463
US
|
Assignee: |
TECHNOLOGY COMMERCIALIZATION
CORP.
45 Tudor City Place Suite 1619
New York
NY
10017
|
Family ID: |
29734690 |
Appl. No.: |
11/970060 |
Filed: |
January 7, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11057776 |
Feb 14, 2005 |
|
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|
11970060 |
Jan 7, 2008 |
|
|
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10178447 |
Jun 21, 2002 |
6953574 |
|
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11057776 |
Feb 14, 2005 |
|
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Current U.S.
Class: |
424/93.44 ;
424/93.4; 424/93.45 |
Current CPC
Class: |
Y02A 50/471 20180101;
Y02A 50/406 20180101; A61K 35/745 20130101; A61P 15/02 20180101;
A23L 7/104 20160801; A23C 9/133 20130101; Y02A 50/30 20180101; A23L
33/135 20160801; Y02A 50/401 20180101; A61K 35/744 20130101; A23L
33/17 20160801; Y02A 50/473 20180101; A61K 35/74 20130101; A61K
35/747 20130101; A23L 19/20 20160801 |
Class at
Publication: |
424/093.44 ;
424/093.45; 424/093.4 |
International
Class: |
A61K 35/74 20060101
A61K035/74; A61P 15/02 20060101 A61P015/02 |
Claims
1. A method for treatment and prophylaxis of a vaginal disease
comprising the steps of providing a hydrolyzed medium with high
acidity of at least 300 T.sup.0, said medium containing at least
one non-pathogenic microorganism and highly concentrated products
of metabolism thereof by fermenting a mixture for about 3-14 days
at about 15-55 degrees C., said mixture comprising at least one
solid food ingredient in small pieces, at least one biocompatible
liquid ingredient and at least one sugar ingredient; and applying
said medium to a patient.
2. The method as in claim 1, wherein said step of providing said
medium including producing said medium by following steps
comprising of: A) providing at least one solid food ingredient
reduced to small pieces; B) providing at least one biocompatible
liquid ingredient containing at least one non-pathogenic
microorganism; C) mixing said solid food ingredient with said
biocompatible liquid ingredient in proportions of about 10-90%
liquid to about 70-5% solid food by weight; D) adding sugar by
mixing it into the mixture at about 0.1-30% by weight; and E)
fermenting the mixture at 15-55 degrees C. until acidity reaches at
least about 300 T.sup.0; whereby obtaining high acidity medium with
high concentration of microorganisms and products of their
metabolism.
3. The method of claim 2, wherein said solid food ingredient is a
plant.
4. The method as in claim 3, wherein said plant is selected from
the group consisting of vegetables, herbs, grains, berries and
fruits.
5. The method as in claim 2, wherein said biocompatible liquid
ingredient is selected from the group consisting of water, juice,
milk, whey, and combination of whey and milk.
6. The method as in claim 2, wherein said non-pathogenic
microorganism is a non-pathogenic bacteria or yeast.
7. The method as in claim 6, wherein said non-pathogenic bacteria
are selected from the group consisting of Lactobacilli,
Bifidobacteria, Streptococci, Pediococci, Leuconostoc, Propionic
and Acetic bacteria.
8. The method as in claim 7, wherein said Lactobacilli are selected
from the group consisting of Lactobacillus Acidophilus,
Lactobacillus Bifidus, Lactobacillus Brevis, Lactobacillus
Bulgaricus, Lactobacillus Delbrueckii, Lactobacillus Casei,
Lactobacillus Cellobiosus, Lactobacillus Fermentum, Lactobacillus
Gasseri, Lactobacillus Helveticus, Lactobacillus Johnsonii,
Lactobacillus Lactis, Lactobacillus Leichmannii, Lactobacillus
Plantarum, Lactobacillus Reuteri, Lactobacillus Rhamnosus,
Lactobacillus sakei, Lactobacillus Salivarius, Lactobacillus
Thermophilus and Lactobacillus Xylosus.
9. The method as in claim 7, wherein said Bifidobacteria are
selected from the group consisting of Bifidobacterium adolescentis,
Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium
cereus, Bifidobacterium infantis, Bifidobacterium lactis,
Bifidobacterium longum, and Bifidobacterium thermophilum.
10. The method as in claim 7, wherein said Streptococci are
selected from the group consisting of Streptococcus lactis,
Streptococcus cremoris, Streptococcus diacetilactis, Streptococcus
thermophilus, and Streptococcus faecium.
11. The method of claim 2, wherein said solid food ingredient is a
solid plant, said step (A) further including providing a high
protein ingredient reduced to small pieces, said step (C) including
mixing said plant, high protein, and liquid ingredients with sugar
in proportions by weight of about 20-85% liquid to 15-80% solids,
and said fermenting step (E) carried out for 3 to 14 days.
12. The method of claim 11, wherein said plant ingredient is
selected from the group consisting of vegetables, herbs, berries,
grains, and fruits.
13. The method of claim 11, wherein said high protein ingredient is
an offal product.
14. The method of claim 11, wherein said high protein ingredient is
a sea product.
15. The method of claim 11, wherein the step (E) of fermenting is
conducted at a temperature of about 35-47 degrees C. for about 3-14
days.
16. The method as in claim 1, wherein said step of providing said
medium including providing said medium with high acidity of at
least 400 T.sup.0.
17. The method as in claim 1, wherein said vaginal disease is a
disease selected from a group consisting of urogenital infection,
cancer of the uterus, cancer of the ovary, and endometriosis.
18. The method as in claim 17, wherein said urogenital infection is
selected from a group consisting of bacterial vaginosis, urinary
and vagina tract infection, yeast vaginitis, gonorrhea, human
immunodeficiency virus infection, herpes, and chlamydia.
Description
CROSS-REFERENCE DATA
[0001] This application is a continuation-in-part application by
the same inventors of the U.S. patent application Ser. No.
11/057,776 filed Feb. 14, 2005 entitled "METHOD FOR TREATING A
MEDICAL CONDITION WITH A HYDROLYZED MEDIUM CONTAINING
MICROORGANISMS", now abandoned, which in turn is a
continuation-in-part of the U.S. patent application Ser. No.
10/178,447 filed Jun. 21, 2002 entitled "METHOD FOR PRODUCING A
FERMENTED HYDROLYZED MEDIUM CONTAINING MICROORGANISMS", now U.S.
Pat. No. 6,953,574.
FIELD OF THE INVENTION
[0002] The present invention relates to a hydrolyzed medium made by
fermentation with non-pathogenic microorganisms and to the process
of manufacturing and use thereof for treatment of vaginal and other
diseases. More particularly it relates to the production of sour
milk based hydrolysate, which includes the fermentation of various
food ingredients and plants using various non-pathogenic
bacteria/yeast ingredients and some food-grade fungi in milk/whey
or water, in its liquid form or solid dried form.
[0003] The medium produced by this method has physiologically
beneficial effects as well as prophylaxis and therapeutic activity
against various diseases, in particular many vaginal diseases such
as urogenital infections and cancers of the ovary and uterus.
BACKGROUND OF THE INVENTION
[0004] The art of fermentation, i.e. the transformation of organic
compounds with the aid of enzymes produced by microorganisms, is
well known. Microbial activity is fairly well understood in the
food industry. The fermentation of food typically refers to the
conversion of sugar into alcohol using yeast under anaerobic
conditions. A more general definition of fermentation is the
chemical conversion of carbohydrates into alcohol or acids.
Fermentation is used widely in the production of alcoholic
beverages, dairy products and some Oriental fermented foods,
especially in tropical climates. Fermentation is also employed as a
form of preservation, creating lactic acid in sour foods such as
pickled cucumbers, kimchi, yogurt and other products. When
fermentation stops prior to the complete conversion of sugar into
alcohol or acids, a stuck fermentation is said to have occurred. A
stuck fermentation is a fermentation which has been stopped before
completion; i.e., before the anticipated percentage of sugars has
been converted by yeast (bacteria) into alcohol or carbohydrates
into carbon dioxide (organic acids). A stuck fermentation may be
caused by: 1) insufficient or incomplete nutrients required for the
yeast (bacteria) to complete fermentation, 2) low temperatures, or
temperature changes, which cause the yeast (bacteria) to stop their
activity prematurely; or 3) the percentage of alcohol (or fermented
end products) has become too high for the particular yeast
(bacteria) selected for the fermentation. In actuality, all
products from the dairy industry are produced by way of stuck
fermentation, where low temperature is used to stop the
fermentation process (for example in yogurt, cheese, etc.), and to
avoid undesirable excess fermentation. The main parameter of
determining the desired endpoint of fermentation used in the diary
industry is titratable acidity, because controlling the extent of
lactic acid production during yogurt and cheese fermentation is
very important. Generally, the production of yogurt takes several
hours (most commonly 2-5 hours), and its titratable acidity is not
high (within 80-100 T.sup.0, or less than 1% lactic acid by
weight). Fruits added into yogurt are not actually fermented during
the process. In fact, steps are taken to prevent fermentation of
such fruit to preserve its taste. Such fruit is added at the very
end of stuck fermentation. Moreover, they are added in dried or
frozen form to avoid increasing titratable acidity. Sometimes,
fruit may be added as artificial flavorings. The yogurt should then
be stored at a low temperature (around 5.degree. C.) to slow down
the physical, chemical and microbiological degradation. However,
fermentation is not stopped at this temperature, it is only slowed
down; therefore the shelf-life of yogurt with live lactic acid
bacteria is less than one month. To increase the shelf-life of
yogurt, the live microorganisms that fermented the yogurt are often
killed by pasteurization. All fermented food products have to be
additionally digested in the gastrointestinal tract after human
consumption.
[0005] About one hundred years ago, Metchnikoff developed a theory
that the ingestion of soured milk could improve colonic microflora
through the reduction of the "auto-intoxication effect" of the
colon. Today, this concept has been improved, and this field is now
known as probiotics and prebiotics, defined as "a live microbial
food supplement that beneficially affects the host animal by
improving its intestinal microbial balance" and "non-digestible
food ingredients that benefit the host by selectively stimulating
the growth or activity of one or a number of bacteria in the
colon". A "synbiotic" is a combination of probiotics and prebiotics
that "beneficially affects the host by improving the survival and
implantation of live microbial dietary supplements in the
gastrointestinal tract by selectively stimulating the growth of,
and/or by activating the metabolism of, one or a number of health
promoting bacteria".
[0006] The bacterial genera most often used in the field of
probiotics are lactic acid bacteria, particularly Lactobacillus sp.
and Bifidobacterium sp., these bacteria being important members of
the gastrointestinal microflora of man and animals. Of all organs
of the body, the vagina is the one where the microbiota is normally
dominated by lactobacilli. Other microorganisms used as probiotics
in humans include Escherichia coli, Streptococcus sp., Enterococcus
sp., Bacteroides sp., Bacillus sp., Propionibacterium sp. and
various fungi.
[0007] The external and internal surfaces of a human body are
covered with bacteria. These organisms are traditionally referred
to as "normal (friendly) flora", or symbionts with commensals.
These friendly bacteria are involved in dynamic bio-film
communications on the skin, mouth, naso-pharyngeal, intestinal and
urogenital tracts, where the appropriate microflora exists. The
human body depends on this friendly microflora: it helps in food
digesting, produces vital vitamins and protects against various
pathogens, especially in the vagina. The mechanisms by which
probiotic microorganisms provide benefits for the internal and
external surfaces of the host are numerous: competing with
pathogens for food, preventing the adhesion of pathogens,
antimicrobial activity, colonization resistance, various immune
effects, adjuvant effect, antimutagenic effects, antigenotoxic
effects, influence on enzyme activity, enzyme delivery and many
others (Sanders (1999) J. Food Technol., 53:67-77).
[0008] Lactobacilli, especially the strains used in probiotics,
manifest antagonism against pathogens (bacteria, yeast, fungi and
viruses). This occurs directly, via competition for limited
nutrients, inhibition of the (epithelial and mucosal) adherence of
pathogens, through the generation of a non-physiologically
conducive acidic environment (i.e., through the production of
lactic or other organic acids) and by the production of various
natural antimicrobial substances (Klaenhammer (1993) FEMS
Microbiol. Rev., 12:39-85; Rolfe (2000) J. Nutrition, 130:
396S-402S). Antimicrobial substances include bacteriocins (Moll et
al., (1999) Antonie van Leeuwenhoek, 76:185-198), low molecular
weight metabolites (such as hydrogen peroxide, lactic and acetic
acids, and other aromatic compounds) and various secondary
metabolites. The latter provides a wide inhibitory spectrum against
many harmful organisms (Saarela et al. (2000) J. Biotech.,
84:197-215). Lactic acid, which possesses antimicrobial properties
by lowering of the pH, has also been shown to permeabilize the
outer membrane of gram-negative bacteria (Alakomi H. L., et al.
(2000) Appl. Environ. Microbiol., 66:2001-2005). It may also act to
potentiate the effects of other antimicrobial substances. Acetic
and propionic acids are also considered to be cell permeabilizers.
Probiotics also have anti-viral properties. It has been proposed
that this is due to their producing an uncomfortable environment
(production of large amounts of acids and other byproducts such as
fatty acids and hydrogen peroxide). Also, although lab-produced
antibiotics have no effect on viruses, acidolin (natural antibiotic
extracted from L. acidophilus supernatant) has both antibiotic and
antiviral properties (effective against polio). Treatment of Herpes
Simplex (oral and genital) has been successful with L. acidophilus
together with L. bulgaricus. One study found that a healthy vaginal
ecosystem (dominated by L. acidophilus) strongly inhibited the
transmission of HIV.
[0009] Now it is a generally recognized fact that probiotics, and
the products of their metabolism, reduce the risk of cancerous
diseases (Sanders, 1999; Hirayama and Rafter, 2000). It has been
proven that probiotics interfere with the display of mutagenic and
genotoxic effects in the colon (Gallaher and Khil, 1999; Reddy
1999), and in other organs (Taper and Roberfroid, 1999; Reddy and
Rivenson, 1993). The inhibitory action of probiotics was observed
at the induction of tumors by cancerogenes (Reddy and Rivenson,
1993; Taper and Roberfroid, 1999), and after the transplantation of
tumors (Taper and Roberfroid, 1999; Kato et al., 1981). Lactic
bacteria and products of their metabolism can inhibit the growth of
cancer cells in culture (Reddy et al, 1973). Moreover, Baricault et
al. (1995) have shown that probiotics can initiate the processes of
differentiation in cancer cells. It is accepted that probiotics are
usually targeted for use in intestinal disorders. The effectiveness
of probiotics has been demonstrated in the prevention and treatment
of a diverse spectrum of gastrointestinal disorders, such as
antibiotic-associated diarrhea, infectious bacterial and viral
diarrhea, etc. Some evidence suggests a role for probiotics in
reducing the risk of colon cancer and the regression of tumors. For
example, U.S. Pat. No. 5,308,615 by DeLoach and U.S. Pat. No.
5,478,557 by Nisbet describe a probiotic used for control of
salmonella. Also, probiotics have been used therapeutically to
lower cholesterol, to reduce blood pressure, treat rheumatoid
arthritis, prevent cancer, and prevent or reduce the effects of
atopic dermatitis, Crohn's disease, constipation as well as
candidiasis and certain genitourinary tract infections such as
bacterial vaginosis, vaginitis, or urinary tract infections. The
immunomodualting action of probiotics is helpful in reduction of
allergic reactions, stimulation of phagocytosis by peripheral blood
leukocytes and secretory IgA, modulation of cytokine gene
expression, and many other immunological effects.
[0010] Probiotic preparations currently on the market appear in
various forms: in dairy products, processed into a product such as
chewing gum, pills, capsules, etc., suspended in milk, freeze-dried
or air-dried (Sanders, Veld (1999) Antonie van Leeuwenhoek, 76:
293-315). They are generally composed of large numbers of one or
more bacterial species that are common constituents of normal
intestinal flora. Fermented milk (yogurt) and cheese are the most
common foods with probiotics. U.S. Pat. No. 6,228,358 by Toba
describes an antioxidation product made from fermented milk. Zhang
describes red rice fermentation products in the U.S. Pat. No.
6,046,022. Other forms of probiotic preparations are freeze-dried
or air-dried and they are available in tablets and in capsules.
U.S. Pat. No. 5,702,927 by Murofushi describes bacteria containing
xanthan gum. In some cases, probiotics have been suspended in an
appropriate milieu for better survival. For example, U.S. Pat. No.
5,908,622 by Barclay describes growing of microflora in
fermentation medium containing certain sodium salts. U.S. Pat. No.
6,294,166 by Hsia describes a method of stabilization of
specifically dried bacterial compositions mixed with specific
nutrients, yeast and soy protein, for long periods of time. Some
authors, for example, U.S. Pat. Nos. 6,203,797 by Perry; 6,080,401
by Reddy; and 5,171,575 by Shibata, used various food/herb
compositions with probiotics, without fermentation, to enhance
medicinal effects.
[0011] PCT patent No. WO 00/75284 by Olshenitsky et. al. describes
a probiotic composition comprising a volatile fraction of a plant
extract prepared by steam distillation and suspended microorganism
such as E-coli. E-coli is not exactly non-pathogenic and may cause
some harm to humans in certain conditions. No fermentation process
of medium ingredients with bacteria is described in arriving at the
end product. Rather, evaporation and condensation is used which
limits the end properties of the product. For example, without
E-coli the product looses its antagonistic activity. Even with
E-coli present, the antagonistic activity is limited because some
pathogens can still grow in the medium during incubation for 24
hours.
[0012] Fermented cultures containing microorganisms can be used in
other industries such as in cosmetics and pharmaceutical industry.
U.S. Pat. No. 6,270,811 by Fregonese describes a composition
containing a microbial culture for skin regenerating and removing
scars and wrinkles.
[0013] Despite their health promoting effects, probiotics have only
demonstrated short-term effects. In the study of the health effects
of probiotics, the incidence and/or duration of acute, short term
diseases, such as diarrhea, are frequently measured. For probiotics
to have their therapeutic effect they should be used in high doses
daily and the duration of their use should be sufficiently long.
The effects of probiotic bacteria on the incidence of diseases with
a protracted etiology, such as cancer or heart disease, have
generally not been measured. Moreover, the effects of probiotics in
life-threatening diseases, such as cancer for example, are doubtful
(Sanders (1999) J. Food Technol., 53:67-77).
[0014] Importantly, attention has been focused on the
microorganisms per se, not their products of metabolism. Lactic
acid fermentation is mainly considered for dairy products. Only in
some oriental foods such as cassaya, mixtures of grains and
legumes, have lactic acid fermentation been used for the
preparation of a variety of foods made from raw materials of plant
and animal origin. Processed food tends to loose a substantial part
of its useful components, ferments for example, as compared to raw
materials.
[0015] U.S. Pat. No. 5,292,511 by Kim describes the lactic acid
fermentation process being used for aloe preservation, and the end
product used as a health-food supplement. The product and process
described in the patent is limited in time (up to 96 hours) and
temperature of fermentation (20-35 degrees C.). At 40 degrees C.
the product is reported to start to decompose. The inventors of the
present invention believe that the fermentation process is not
complete from the point of view of the instant invention.
[0016] U.S. Pat. No. 4,298,620 by Hagiwara proposes a fermentation
process for obtaining a fermented tear grass product combining a
water extract of tear grass with a Lactobacillus strain, and foods
and feeds comprising that product. This patent is incorporated
herein in its entirety. Importantly, one critical step in the
process as described in this patent is heating of the tear grass
before fermenting it. In our opinion, this step effectively damages
all useful ferments contained in the grass and significantly
reduces its effectiveness. Also, since the number of bacteria is
not reduced at the end of cultivation, the acidity of the end
product (as measured by concentration of lactate) is low at about
0.7 to 3%. Another limitation is the typical addition of sugar at
the end of cultivation. Finally, a heat sterilization process at 80
degree C. for 40 minutes effectively destroys all live
microorganisms and active ingredients, ferments for example.
[0017] Other fermentation patents of interest include U.S. Pat.
Nos. 5,219,597 by Mok; 5,700,684 by Ehret; 6,156,320 by Izvekova;
5,556,785 by Kishida; 5,747,020 by Rutherford; 4,407,828 by
Raccach; 3,963,835 by Gryczka; 4,018,650 by Busta; 4,528,199 by
Moon; 4,579,740 by Matrozza; 4,897,350 by El-Megeed; 4,749,652 by
Robinson; 4,816,267 by Oka; 4,855,147 by Yokota; 4,579,739 by
Darbyshire; 4,664,919 by Yan; 4,770,882 by Ingouf; and 3,944,676 by
Fridman. They depict mostly various fermentation processes that are
somewhat similar to the subject of the invention but in most cases
these processes are short-term or carried out at low temperatures
in solid phase and therefore incomplete from the point of view of
the present invention in that they describe various methods of
stuck fermentation, and so the end by-products of which have to be
additionally digested in gastrointestinal tract after human
consumption. Digestion in humans, as in other animals, is the
process by which food containing nutrients such as proteins, fats,
and carbohydrates is eaten and broken down into its components,
i.e. the breakdown of biodegradable material. These components are
absorbed by the blood from the small intestine and dispersed
throughout the body for use by various organs and cells. Salivary,
gastric and other types of enzymes initiate and facilitate
digestion in the gastrointestinal tract. Beneficial bacteria, which
live in symbiosis (ecto-symbiosis) in the gastrointestinal tract of
humans and other mammals, are very important in preparing the food
for absorption and in the assimilation of nutrients.
[0018] Probiotic bacteria produce enzymes that: [0019] 1) break
down proteins into amino acids and peptides, [0020] 2) break down
fats and complex sugars, and [0021] 3) improve the
adsorption/bio-availability of calcium and other minerals (Fadda et
al. (2002) J. Food Sci., 67:1179-1183; Hugenholtz et al. (2002)
Antonie van Leeuwenhoek, 82:217-235). Lactobacilli produce lactase,
helping those that are lactose-intolerant to digest lactose.
Lactobacilli are able to predigest proteins, fats, or carbohydrates
in such form that is more readily absorbed and digested in mammals.
Some strains can produce almost all B vitamins, including niacin,
biotin, B6, B12, and folic acid. Such bacteria, which live in our
large intestine, can be considered as a part of the digestive
system, because they assist in breaking down food into its
most-elementary form, such as amino acids, glucose and fatty acids.
Soluble fibers, which can be found in fresh and dried fruit,
vegetables, oats, legumes and seeds, are fermented by bacteria
within the large intestine and can assist in maintaining colon
health and increasing mineral absorption. Some vegetables may
contain complex carbohydrates, which the human digestion system
cannot digest properly, but gastrointestinal bacteria help the host
digest it. Bacteria produce certain enzymes that higher-order
organisms do not produce in order to degrade certain biomolecules.
Natural resistant starch being insoluble is fermented in the large
intestine, and is considered as a prebiotic fiber. The digestion
process begins with bacterial hydrolysis of the input materials in
order to break down insoluble organic polymers, such as
carbohydrates and make them available as food for other bacteria.
Acidogenic bacteria then convert the sugars and amino acids into
carbon dioxide, hydrogen, ammonia, and organic acids. For example,
Lactobacilli are capable of turning milk sugar into lactic acid.
Lactic acid-producing bacteria may be subdivided into two groups:
homo-fermentative (produce more than 85% lactic acid from glucose)
and hetero-fermentative (produce only 50% lactic acid and
considerable amounts of ethanol, acetic acid and carbon dioxide).
Acetogenic bacteria then convert these resulting organic acids into
acetic acid, along with additional ammonia, hydrogen, and carbon
dioxide. Finally, Methanogenic bacteria are able to convert these
products to methane and carbon dioxide. The mechanisms accounting
for the composition of the gut flora and its genesis are
incompletely understood. Some results suggest that the normal
symbiotic flora are essential to homeostasis in the gut and may
promote intestinal development that may serve to further enhance
the host's nutrition (Sears (2005) Anaerobe, 11:247-251;
Rakoff-Nahoum (2004) Cell, 118:229-41). Such symbiotic relationship
is good for the host organism and there are also benefits for
symbiotic microflora. Such benefits include food decomposition,
metabolic by-products of fermentation of saliva and
gastrointestinal food, constant temperature for bacterial ecosystem
and others. For example, Lactobacilli grow more and produce more
extracellular metabolites after the addition of yeast extract and
peptone as well as hydrolysed proteins such as whey protein
hydrolysate to the milk, because milk lacks some vitamins, peptides
or amino acids that are essential for bacterial growth (Ruiz-Barba,
Jimenez-Diaz (1994) J. Appl. Bacteriol., 76:350-355; Morishita et
al. (1981) J. Bacteriol., 148:64-71. There is even more rich food
for lactobacilli in the gastrointestinal tract. One goal of this
invention is to demonstrate that probiotic bacteria are capable of
digesting food without the presence of a host, so-called "external
digestion". As with human digestion, the end-products of such
external digestion are very similar, regardless of what types of
input food products are processed. After digestion by a healthy
human, the quantity and quality of the excrement is little
different, regardless of what is eaten. Moreover, the products of
such "external digestion" of the present invention are the result
of a deep (or near-complete) fermentation process, given that
additional degradation of biodegradable food material is not
observed at the end of such fermentation. In fact, such food
material is now in its most-elemental form, virtually the same as
that transported in the bloodstream (after digestion in the
intestines) Therefore, such hydrolyzed food material of the present
invention can be utilized by the host macro-organism, not only
through the gastrointestinal tract, but also through the skin,
colon and vagina, where direct external application can be made.
This application to the host macro-organism is very beneficial for
it, because it locally stimulates appropriate organs through
stimulation of calcium signaling in the cells (Sobol (1995) Gen.
Physiol. Biophys., 14:293-303; Sobol et al. (2005) Neurophysiology,
37:284-293). Moreover, when locally applied to the skin, it
stimulates immune reactions, which are very important for patients
with life-threatening diseases. Such deep fermentation makes our
product quite different from other fermented food products like
yogurt, cream, cheese and others, which are produced by stuck
fermentation (incompletely digested) and may be utilized only
through the gastrointestinal tract. The high concentration of
lactate in the present invention has many benefits, including it's
role in the preservation of our product under storage, inhibiting
the growth of various pathogens, so that it may remain sterile
(except for probiotic bacteria) for a long time in unsterile
environments. The preferable acidity is above 500 T.sup.0, because
such concentration of lactate is better at suppressing pathogens
effectively. However, high acidity (above 800 T.sup.0) may be toxic
to some extent.
[0022] One probable reason for limited effectiveness of probiotics
in general is because of poor binding of the active microorganisms
to the internal linings or external surface of the human body.
Bacteria, especially in the state of freeze-dried suspension, have
only limited time to develop a bond with the host. It takes several
hours for the bacteria to become active after being consumed.
Therefore, the bacteria are frequently expelled by natural
processes such as digestion without allowing it to bond to the
intestines and to produce enzymes, vitamins, amino acids, organic
acids and other products of their metabolism. It is these metabolic
products that represent the ultimate goal of the application of
microorganisms. Live microorganisms might have a better chance to
remain on the surface and tissue lining and attach thereto.
[0023] The need therefore exists for a medium containing live
microorganisms as well as their metabolic products in high
concentrations. Its application for humans is believed to be more
effective and provide long-term benefits than the presently known
suspensions of such microorganisms mostly in inactive state, even
consumed in a high concentration.
SUMMARY OF THE INVENTION
[0024] It is an object of the present invention to provide a
composition for a biologically benign medium containing
non-pathogenic microorganisms and their metabolic products, such as
enzymes with high proteolytic activity, vitamins, amino acids, low
molecular weight proteins, organic acids, microelements and others
as well as to use this medium for prophylaxis and treatment of
vaginal diseases.
[0025] It is another object of the invention to provide a fermented
medium allowing the microorganisms to remain alive in active state
so that better conditions are created for attachment thereof to the
appropriate tissue of the host.
[0026] A further object of the present invention is to provide a
medium of high acidity, at least above 3% of lactate concentration
(>300 T.sup.0 or degrees Turner), to promote higher vitality of
those microorganisms that survive in the process of natural
selection in a harsh for them acidic environment. It is noted here
that lactate also plays a role of a preservative for the medium of
the invention.
[0027] It is another object of the invention to provide such medium
based on raw natural fish, animal, and plant products not subjected
to heavy food processing or application of heat to retain and
preserve original ferments and other useful ingredients.
[0028] It is another object of the present invention to provide a
method for producing such medium. A further object is to provide a
new process of deep fermentation of the medium with appropriate
microorganisms to cause production of metabolic products useful for
human beings, so-called "external digestion".
[0029] It is a further object of the present invention to provide
methods of use of the medium of the invention in medicine, food,
cosmetics, and other industries.
[0030] It is a further yet objective to provide a medium capable of
producing long-term therapeutic effects on a human being or an
animal.
[0031] A most beneficial application of probiotics according to the
present invention is in woman health, because the normal vaginal
microbiota is dominated by lactobacilli, especially Lactobacillus
crispatus, Lactobacillus jensenii, Lactobacillus iners and
Lactobacillus gasseri (Falagas et al. (2007) Clin. Microbiol.
Infect., 13: 657-664). In fact, the vaginal microbial flora may
play a role in maintaining human health (Sobel, (2000) Annu. Rev.
Med., 51: 349-356; Cadieux et al. (2002) JAMA, 287:1940-1941).
There is strong evidence that the absence of vaginal lactobacilli
is associated with the development of bacterial vaginosis
(Alvarez-Olmos et al., (2004) Am. Sex. Trans. Dis. Assoc., 31:
393-400). Lactobacillus species form a barrier population that
protects the host from pathogen colonization by mechanisms that
include adhesion to epithelial surfaces, self-aggregation and
co-aggregation. There is much in vitro and in vivo data showing
inhibition of vaginal pathogens by appropriate lactobacillus
strains and by their cell-free culture supernatant (Burton (2003)
Appl. Environ. Microbiol., 69:97-101; Reid et al. (2003) Nutraceut.
Food, 8:145-148 and FEMS Immun. Med. Microbiol. 35: 131-134; Atassi
et al. (2006) FEMS Immunol Med. Microbiol., 48: 424-432; Falagas et
al. (2007) Clin. Microbiol. Infect., 13: 657-664; U.S. Pat. No.
6,479,051 by Bruce, et al.; U.S. Pat. No. 6,468,526 by Chrisope and
many others). The mechanism(s) underlying the antagonistic activity
of Lactobacillus strains appear to be multifaceted, and include the
production of hydrogen peroxide, lactic acid, and antibacterial
compounds including bacteriocins or bacteriocin-like molecules,
non-bacteriocin molecules, and non-lactic acid molecules (Servin
(2004) FEMS Microbiol. Rev., 28: 405-440). The production of lactic
acid by lactobacilli is primarily responsible for the low vaginal
pH (below 4.5) and is one of the important factors for the
inhibition of the growth of uro-pathogens (Tomas et al., (2003) J.
Med. Microbiol., 52, 1117-1124). It should be noted that the
killing activity exerted by the compounds(s) present in culture
supernatant of some probiotic strains is potentiated in acidic
conditions (Atassi et al. (2006) FEMS Immunol Med. Microbiol., 48:
424-432). Nevertheless, for the majority of known selected
probiotic Lactobacillus strains, the compound(s) exerting killing
activity against uro-pathogenic, entero-virulent or
vaginosis-associated bacterial pathogens have not been
characterized (Servin, (2004) FEMS Microbiol. Rev., 28: 405-440).
In actuality, the presence of lactobacilli does not necessarily
exclude potential pathogens from the vagina, as there is a constant
battle between friendly and pathogen bacteria there involving host
defenses (Saunders et al. (2007) Colloids and Surfaces B:
Biointerfaces 55 (2007) 138-142). Administration of lactobacilli
and products of their metabolism shifts this equilibration forward
to healthy state, i.e. lack of symptoms and signs of disease, and
the regained dominance of lactobacilli. A frequent source of
pathogens for urinary and vaginal tract infections in women is the
intestinal tract (Sanders (2000) J Nutr., 130:384 S-390S).
Therefore, oral administration of Lactobacilli may provide a
therapeutic effect for such problems in women. However,
intra-vaginal or oral plus intra-vaginal application is likely to
be even more efficient.
[0032] In accordance with the present invention, a new symbiotic
multi-component fermented hydrolyzed medium is provided with a
broad spectrum of antibacterial, antiviral and anti-fungal
properties, and antagonistic activity against Protozoa. The medium
is produced with non-pathogenic microorganisms, and has a high
concentration of aromatic organic acids such as lactate, acetate,
propionic, and other organic acids as metabolic end products of the
fermentation process. The medium of the invention contains live
non-pathogenic microorganisms in low concentrations and the
products of their intensive metabolism, thereby keeping
microorganisms in their most active alive condition. It is also
believed that the bacteria that remain alive have more vitality,
due to the natural selection of those that can survive in the
environment of high acidity. Non-pathogenic microorganisms genera
used are Lactobacilli, Bifidobacteria, Acetic and Propionic
bacteria, yeasts and food-grade fungi.
[0033] According to the invention, the fermentation process for
obtaining sour-milk hydrolyzed medium is as follows. Initial
ingredients include certain raw or dried vegetables, fruits,
berries, offal, fish, eggs, plants, herbs, mash, sprouted grains
and beans, aquatic plants, products of beekeeping, sea products,
mushrooms, proteolytic ferments, chemicals and various types of
sugar in the appropriate amounts. The ingredients are mixed in
predetermined proportions and fermented with non-pathogenic
bacteria or yeasts and certain food-grade fungi in milk or whey.
Fermentation can also take place in water or another appropriate
biocompatible liquid. The fermentation process takes 3-14 days
(preferably 5-10 days) at 15-55 degrees C. (preferably 32-47
degrees C.) and can be carried out both aerobically and
anaerobically. Typically, sour milk hydrolyzed medium includes
about 10.sup.5 to 10.sup.6 live bacteria/yeasts cells per 1.0 g of
product, and comprise 1 to 30 percent by weight of protein, all
essential amino acids (resulting from the partial proteolysis of
proteins during culturing), organic acids, microelements and
vitamins. The acidity of the final product is 300 to 900 T.sup.0,
preferably between 500 to 800 T.sup.0, which corresponds to lactate
concentration of between about 4.5% and 8%, and pH ranges from 1.5
to 6.5, preferably from 3 to 4. There is no heat processing or
pasteurization used in preparation of the medium.
[0034] The medium of the invention includes only food products, is
not toxic and quite safe, and can be successfully utilized in
high-risk patients, such as the elderly, hospitalized and the
immunocompromised, including AIDS patients. No side effects were
observed in babies or pregnant women in our studies.
[0035] The efficacy of the medium of the invention is comparable to
modern pharmacological drugs (antibiotics, and antiviral and
anti-fungal compounds). Moreover, this medium is shown to be
effective against life-threatening diseases, such as cancer,
tuberculosis, HIV/AIDS, pneumonia and others, where traditional
pharmacological drugs failed. No drug interaction was observed
between pharmacological drugs and the medium of the present
invention. On the contrary, the medium reduced considerably the
side effects caused by toxic pharmacological drugs.
[0036] The medium was found to possess a broad spectrum of
therapeutic potential (the application was not limited to only GI
tract) including reduction of DNA damage of the host cells.
Boosting the immune system, immunomodulation, normalization of the
number and function of blood cells, especially lymphocytes, are the
most pronounced effects of this medium. These effects appear to be
mainly caused by the end products of bacteria's metabolism.
[0037] Methods of administration of medium of the invention, in
addition to accepted oral and intravaginal administration, include:
1) the external application to the skin as a bandage to the
effected organs or coating the body (rubbing it into the skin),
mostly the trunk and lymph nodes, 2) inhalation, 3) administration
rectally via a retention enema, 4) dripping into the nose and ears,
5) intravenous injections for reducing infections and/or for
intravenous nutrition, and 6) intraperitoneal injections for
reducing infections.
[0038] The above and other objects, aspects, features and
advantages of the invention will be more readily apparent from the
description of the preferred embodiments thereof taken in
conjunction with the appended claims.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0039] The first most important and unique aspect of the present
invention is providing temperature and time conditions for the
process of fermentation which ensure that at the end the
fermentation is deep and complete. We found that generally at a
point of about three days into the process of fermentation, most
pathogens are destroyed/replaced. The medium of the present
invention has to be fermented for at least 3 and preferably 5 to 14
days at a temperature of at least 15 and preferably 35 to 55
degrees C. to make sure that no pathogens are present. Another
objective for this long time and higher temperature (as opposed to
what is described in the prior art) is to make sure that
microorganisms have adequate conditions to release products of
their metabolism into the medium. By the end of the process, the
medium has high acidity (generally, from 300 to 900 T.sup.0), low
pH (from 1.5 to 6.5), high concentration of the metabolic products
and relatively low concentration of microorganisms (from 10.sup.5
to 10.sup.6 cells/ml) as opposed to the preparations described by
others.
[0040] In the most basic form, the medium of the invention can be
prepared following these steps: [0041] 1. Provide a food product of
plant nature, wash it and cut into small pieces (dimension of
pieces can vary from about 1/2 millimeter to 4 centimeters). Juice
extractor may be used for such purpose. The smaller the pieces are,
the better hydrolysis will be achieved. A wide variety of food or
plant products may be used, for example most fruits, vegetables,
berries and herbs. [0042] 2. Provide a biocompatible liquid such as
water, juice, etc. Most preferably, whey or milk or their
combination may be used. Lemon, orange, or grape juice is another
preferred biocompatible liquid. [0043] 3. Mix the food ingredient
with the liquid ingredient in proportions of 10-90% liquid to 70-5%
food by weight. [0044] 4. Add sugar to this mixture at 0.1-30% by
weight. Mix thoroughly and place in a thermostat for about 5-14
days at 32-55 degrees Celsius.
[0045] Ambient microorganisms cause fermentation to proceed. For
whey and milk, it is a naturally present Lactobacillus. Optionally
and to better control fermentation, appropriate strain of
microorganism can be specifically added, Lactobacillus bulgaricus
for example.
[0046] The fermentation endpoint is determined by measuring the
acidity in Terner degree. A satisfactory acidity for the medium is
between 300 and 900 T.sup.0, and pH ranges between 1.5 to 6.5.
[0047] The strength and quality of the fermented medium of the
invention depends on the number and nature of various ingredients
and their proportions. Another unique aspect of the invention is to
provide ingredients with high concentration of proteins such as
tissue and organs of fish, poultry, animals and others. Offal
ingredients are most preferred. As such, the second preferred
method of producing the medium of the invention comprise providing
at least one food/plant ingredient such as a vegetable, fruit,
berry or herb as described above and one high protein ingredient
such as an offal component, mushroom, sea product (fish, mussel,
plankton for example), egg or nut. Proportion for plant with high
protein ingredient and liquid is ranging from 15-80% solids to
20-85% liquid. In comparison to the first embodiment, the
processing parameters may be opened up somewhat without
compromising the completeness of the fermentation process and
achieving high acidity at the end. The temperature range in this
case is 15 to 55 degrees Celsius and the time range is 3 to 20
days. It is still preferred to maintain higher temperature of 32 to
47 degrees Celsius and ferment the medium for at least 5 days so
the acidity reaches a level above 300 T.sup.0.
[0048] According to the third preferred embodiment and to achieve
maximum strength, the composition of and preparation process for
the medium of the invention are described in the following steps:
[0049] 1. Provide for sprouting of at least one grain such as rye,
lentil, wheat or barley, and beans for 2-6 days at 20-30 degrees C.
in humid air. At the end of this period, grains can be optionally
seeded with food-grade fungi such as Aspergillus niger and/or
Aspergillus orizae to increase proteins concentration. [0050] 2.
Inoculate a biocompatible liquid such as sterile milk or whey for 1
to 24 hours at 20-35 degrees Celsius with at least one of selected
non-pathogenic microorganisms such as bacteria/yeasts. The number
of live bacteria at this point is in the general range of from
about 10.sup.7 to 10.sup.9 per ml of liquid, and pH is maintained
close to neutral. Optionally, fermentation in water or juice can
also be used.
[0051] Examples of non-pathogenic bacteria that can be used for the
medium of the invention include, but not limited to, all strains of
Lactobacilli, Bifidobacteria, Streptococci, Pedicocci, Leuconostoc,
Propionic and Acetic bacteria. The yeast is Brewer's or Baker's
yeast, which is added in active or non-active form (dried,
autolyzed, hydrolyzed or extract). These non-pathogenic
bacteria/yeasts can be alternately added without inoculation,
immediately after the mixing of the various ingredients according
to step 4. In this case however, fermentation will require more
time.
[0052] Preferred strains of lactobacilli to be used in the medium
of the invention include, but not limited to, lactobacillus
acidophilus, lactobacillus bifudus, lactobacillus brevis,
lactobacillus bulgaricus, lactobacillus delbrucki, lactobacillus
casei, lactobacillus cellobiosus, lactobacillus fermentum,
lactobacillus gasseri, lactobacillus germentum, lactobacillus
helveticus, lactobacillus johnsonii, lactobacillus lactis,
lactobacillus leichimanii, lactobacillus plantarum, lactobacillus
reuteri, lactobacillus rhamnosus, lactobacillus sake, lactobacillus
salivaroes, lactobacillus thermophilus and lactobacillus
xylosus.
[0053] Preferred strains of Bifidobacteria include Bifidobacterium
adolescentis, Bifidobacterium bifidum, Bifidobacterium breve,
Bifidobacterium cereus, Bifidobacterium infantis, Bifidobacterium
lactis, Bifidobacterium longum, and Bifidobacterium
thermophilus.
[0054] Streptococci strains to be used include preferably
Streptococcus lactis, Streptococcus cremoris, Streptococcus
diacetylactis, Streptococcus thermophilus, and Streptococcus
faecium.
[0055] Preferred yeast includes Saccharomyces boulardii,
Saccharomyces cerevisiae, and Saccharomyces lactis.
[0056] Propionic bacteria strain is preferably propionibacterium
shermanii.
[0057] Pedicocci strains that may be used in the present invention
include Pediococcus cerevisia, Pediococcus acidilactici and
Pediococcus pentosaceus.
[0058] Leuconostoc strains include Leuconostoc cremoris,
Leuconostoc dextranicum, and Leuconostoc mesenteriodes. [0059] 3.
Provide other ingredients as specified below to include at least
one type of dried or preferably fresh vegetables, fruits, berries,
offal, and herbs, all thoroughly washed with water in addition to a
product of beekeeping, mash, and proteolytic ferments. All
products, including sprouted grains, should be
homogenized/pulverized or mechanically processed, for example
through juice extractor. In case of dried products, their amount
should be 3-5 times less by weight, they should be soaked in an
appropriate volume of water for some time (for minutes or hours
depending on type of product) to produce the same quantity of mass
as when using fresh ingredients.
[0060] Vegetables preferably used are of leaf and root types e.g.
various cabbages, beets, rutabaga, carrot, pumpkin, spinach, beet,
watermelon, melon, peanut, artichoke, eggplant, pepper sweet,
asparagus, and tomato. Fruits to be preferably used are apples,
pears, kiwi, plums, citrus, apricots, grapes/raisins, mango, guava,
bananas, biwa, cornel, fig, cherry plum, quince, peach,
pomegranate, avocado, pineapple, date, papaya. Berries preferably
include raspberry, bilberry, guelder rose, dog rose, ash berry (red
and black), currant (red, black, and white), sea-buckthorn berries,
gooseberry, schizandra, blackberry, cowberry, bird cherry,
cranberry, sweet cherry, cherry, and strawberry. Preferred herbs
and their roots are ginseng, celery, parsley, dill, dandelion,
nettle, ginseng, and spinach. Preferred high protein products are
offals including spleen, kidney, heart, liver, brains, maw, and
stomach as well as mushrooms, sea products (fish, mussel, plankton
for example), eggs or nuts. Preferred products of beekeeping are
propolis, honey, royal jelly, and pollen of flower. [0061] 4. Mix
the preferred composition of the above ingredients as follows: 25
to 80% by weight of inoculated milk or whey, 1 to 30% by weight of
vegetables, 1-20% by weight of fruits, 1-20% by weight of berries,
1-15% by weight of herbs, 1-30% by weight of high protein
ingredients, 0.1-5% by weight of products of beekeeping, 1-10% by
weight of sprouting grains and beans, 1-15% by weight of mash,
0.1-1.0% by weight of proteolytic ferments (pepsin or alike).
[0062] 5. Provide specified amount of sugar, 0.1-30% by weight, and
thoroughly mix into the medium. Preferred types of sugar include
glucose, fructose, sucrose, mannose, maltose, galactose, raffinose,
corn syrup, lactose or other mono-, di- or polysaccharides, which
can be utilized by Lactobacilli or Bifidobacteria. These sugars can
be used both in combination and alone. [0063] 6. Provide at least
one of the following chemical compounds: potassium, sodium,
magnesium, calcium, trace of cobalt, trace of manganese, and
alcohol. The chemical compounds should be dissolved in water and
added to the mixture. Optionally, horns and hoofs can be used, but
this might worsen the taste of the product. [0064] 7. Ferment all
of the above ingredients aerobically or anaerobically for 3-20 days
at 15-55 degrees C. It is preferred to ferment the mixture for 5-10
days at 32-47 degrees C. [0065] 8. After fermentation, separate the
liquid (for example by filtering of with a centrifuge), which
constitutes the desired sour milk hydrolyzed medium of the
invention. The number of live bacteria in the liquid medium at the
end of fermentation is about 10.sup.5 to 10.sup.6 per one gram of
liquid. The removed sediment contains all the useful ingredients as
well and the same bacteria and can be used as feed supplement for
human consumption and in animal husbandry. The sediment can be
alternatively lyophilized at room temperature and stored for later
consumption.
[0066] The resulting products, sour milk hydrolyzed medium and
sediment, can be stored at room temperature for several weeks. When
refrigerated at 2-15 degree Celsius, it can last for up to several
years without deterioration. No preservatives need to be added.
[0067] The acidity of the final liquid medium product is 300 to 900
T.sup.0, preferably between 500 to 800 T.sup.0, which corresponds
to lactate concentration of between 4.5% and 8%, and pH ranges from
1.5 to 6.5, preferably from 3 to 4.
[0068] The products of the suggested fermentation process are not
toxic and have application in medicine, food industry,
biotechnology, veterinary, animal, poultry, and fish husbandry,
athletic sport as a food supplement, and/or therapeutic drugs and
can be used as a prophylactic agents against diseases. The medium
was observed to be non-toxic, even for challenging groups of
patients such as the elderly, hospitalized and the
immunocompromised, including AIDS patients, expecting mother and
babies. The most pronounced effects of the medium of the present
invention include immunomodulation, improving physiologic function
at the cellular level, for various organs and the entire body. The
medium of the invention has a broad spectrum of antimicrobial
activity with the ability to destroy and/or inhibit growth of many
different species of pathogenic microorganisms.
[0069] Methods of administration of the medium of the invention
depend on the specific condition. For general application, oral
administration is most useful. Other applications include: 1) the
external transcutaneous application as a bandage soaked in the
medium placed over the skin above effected organs or coating the
body (rubbing it into the skin), mostly the trunk and particularly
lymph nodes, 2) inhalation, especially for breathing disorders, 3)
administration rectally via a retention enema, 4) dripping into the
nose and ears, 5) intravenous injections (after removal or
microorganisms via known means such as through a filter) for
reducing infections and/or for intravenous nutrition, 6)
intraperitoneal injections for reducing infections, and 7)
intravaginally via a soaked tampon.
[0070] A proper dosage of the medium and sediment of the present
invention in humans depends upon the particular needs. In case of a
life threatening condition, the application of the medium of the
invention should be the most aggressive and combine several
possible routes of administration. As an example, an AIDS patient
should take the medium of the invention orally 2-3 times a day at
any time in the amount of about 1 to 2 ml/kg of body weight in
addition to coating the body with the medium as much skin area as
possible, especially lymph nodes, 1 to 2 times a day, in the
morning and at night. In case of liver, kidney, pancreas, etc.
intoxication caused by HAART, skin bandages soaked with the medium
should be placed over the skin above effected organs 1-4 times a
week. In case of lung problems, additional inhalation of the medium
on a daily basis is needed as well. Our evaluation showed that all
lung infections in AIDS patients disappeared within 1-3 weeks.
Additionally, rectal administration of the medium, typically with
oil, should be used 3-5 times a week. Women should use intravaginal
tampons wetted with the medium of the invention at least 2-4 times
a week for up to several hours at a time. Our studies demonstrated
that as a result of such intensive therapy, opportunistic
infections resolved mostly within 2-8 weeks, CD4 increased by 20-30
cells/month, accompanied with increasing CD8 and ratio CD4/CD8.
After 1-4 months of such therapy, it can be interrupted for 1-3
weeks or the doses and/or number of methods of administration can
be reduced. For a less serious condition, oral administration is
generally enough.
[0071] The following are examples of specific use of the medium of
the invention. All patients were diagnosed and treated in Russian
hospitals.
[0072] CASE 1. Patient: women 42 years old. DIAGNOSIS (2001):
Fibrous cavernous tuberculosis of left lung in phase of
infiltration. The infiltration is massive with many cavities of
disintegration. Some planting of right lung. Tuberculosis of
throat. Intoxication and pneumorrhagia (blood streaked sputum).
Chronic pyelonephritis (since 1980). MT (+), the process was
complicated with caseous pneumonia as independent condition,
tuberculosis intoxication, exhaustion, toxic anemia and lung-heart
insufficient of the second stage.
[0073] BASIC MEDICAL TREATMENT (B.M.T.) with antibiotics:
streptomycin, isoniazid, rifampin, pyrazinamid, ethambutol and
ofloxacin for some months. Resulted in the progression of the
tuberculosis process. She could not stand up from her bed.
[0074] MEDICAL PROGNOSIS: not favorable.
[0075] APPLICATION OF THE MEDIUM OF THE INVENTION. The product of
the present invention was administered (oral, inhalation and
applying to body at lymph nodes). Her blood test results improved
within 5-7 days, immunology improved within 1 month, x-rays
improved within 2 months, and body temperature (fever) started
normalizing within 2-3 weeks. After 1 month, she decided to stop
antibiotic administration. By the 11th month of administration of
the product of the present invention, her weight had increased by
22 kg. Her left lung was completely auscultated. She is living now
(2007) with additional improvement of her left lung (pneumatization
of damaged lung tissue). It should be noted that her left lung
would not have recovered without administration of the product of
the present invention.
[0076] CASE 2. Patient: women 73 years old. DIAGNOSIS (2001):
caseous pneumonia of left lung in phase of infiltration and
planting of right lung. MT(+), tuberculosis intoxication,
cardiac-lung insufficient of second/third stage. Accompanying
diseases: myocardiostrophy, atherosclerosis of coronary and
peripheral arteries and aorta. Atherosclerotic cardiosclerosis.
[0077] BASIC MEDICAL TREATMENT (B.M.T.) with antibiotics: rifampin,
streptomycine, pyrazinamid, phthivazidi, ethambutol administered
for several months resulted in negative dynamics of the
tuberculosis process. In spite of some normalization of blood
tests, dyspnea, coughing, weakness, and intoxication were
increasing. X-ray results were getting worse. The patient was
hardly moving and was very exhausted. Some quantity of locuses in
the right lung reduced, but in the left lung they increased
considerably.
[0078] MEDICAL PROGNOSIS: not favorable.
[0079] APPLICATION OF THE MEDIUM OF THE INVENTION. The product of
the present invention was administered for the most part externally
to her body (greasing her chest as frequently as possible 10-15
times a day and her lymph nodes 1-3 times a day, and after around
four weeks she could orally ingest the product of the present
invention). All symptoms of intoxication in her blood tests (in
erythrocytes) disappeared within 2-3 weeks, x-ray results improved
within 2 months, MT(-). Patient physical state improved
considerably, she became much more active. Her weight increased by
approximately 10 kg and she could fully take care of herself. She
was discharged home in four and half months after our product
administration. (B.M.T.) was not interrupted.
[0080] CASE 3. Brief history. Patient, male, aged 70 years. The
final diagnosis of the main pathology: cancer of oesophagus.
Operation: resection of oesophagus. Risk of operation III-IV stage.
Complication: suppuration of the surgical wound on the neck and
failure of anastomosis. The wound healed with the second operation.
The patient was discharged home with an improvement in condition.
The postoperative period was 53 days. Description of the disease.
The patient complains of the presence of a tumour-shaped mass and
pain in the area of the right sternoclavicular joint. He
experiences difficulties in passing food along oesophagus. He was
previously operated 19 years ago for cancer of oesophagus, with a
presternal replacement of oesophagus with colon. In the course of
the surgery there was clinical death on the operational table.
Histology No. 44-47: an area of oesophagus with a narcotising
tumour involving all layers of the oesophageal wall. Final
diagnosis: squamous cell carcinoma. Brief description of operation,
during the operation a tumour of oesophagus is revealed. Its lower
edge is located on the level of the former oesopageal-colonic
anastomosis, while the upper edge is on the neck. The tumour
measures about 5 cm. Two soft grey lymph nodes are present in the
perioesophageal tissue. The decision was made to do resection of
the lesioned area. An end-to-end oesophageal-colonic anastomosis
was performed. After the operation, his surgeon noticed that he had
observed multiple metastases in the patient's chest cavity and
advised that the patient was to live as little as 2, maximum 4
weeks. Therefore, no gastrostoma seemed to be done. The
postoperational period was complicated with suppuration of the
wound and failure of the anastomosis. Nutrition was performed only
intravenously: one litre of 40% glucose (several times), casein,
and alcohol. Three days after operation significant pus discharges
with an odour were observed.
[0081] APPLICATION OF THE MEDIUM OF THE INVENTION. The patient
asked for the administration of the products of the present
invention to help him. He started greasing his skin with it around
the bandage and started rectal administration of our product with
different juices, predominantly blueberry juice. As a result, the
wound suppuration began to decrease, and shrank significantly.
Eight days after operation, hemotransfusions started. On the
37.sup.th day after surgery, the swallowing reflex appeared. By the
50.sup.th day, the patient gained in weight by 2.5 kg. On 53.sup.rd
day, he was discharged in a satisfactory state. The patient
continued administration of products of the present invention at
home for the next month. He died 7 years after the operation (at
the age of 77) from a disturbance of the cerebral circulation.
During this period, he was able to completely take care of himself.
His blood and urine analyses were without pathology.
Survival and Index of Proliferation
[0082] Human chronic myeloid leukemia cells (line K-562, bank of
cellular cultures at the Institute of Cytology, Russian Academy of
Science, St.-Petersburg, Russia) were used in experiments in vitro.
Cells were cultured in a 96-hole stripe with DMEM/F12 (1:1), with
15 mmol/l HEPES and with 10% bovine serum. On the first or second
days of cultivation, cells were stained with trypan blue and the
survival and index of proliferation were evaluated using a
microscope. The number of cells analyzed was 250-400 thousand in
each experiment. The product of the present invention in
experiments in vitro was centrifuged at 5000 rpm for 20 minutes,
then filtered through Millipore filters (0.22 mkm) to remove
fermenting bacteria. The product was diluted 1/40 (2.5%) and pH was
adjusted to 7.4 with NaHCO.sub.3. Results of three independent
experiments on the survival and index of proliferation of myeloid
leukemia cells are presented in Table 1. In these experiments, the
pH of the product was neutralized and a dilution of 1:40 was
used.
[0083] It can be seen from Table 1 that the product of the present
invention significantly reduced the index of proliferation (by
almost two times, p<0.01) of human chronic myeloid leukemia
cells (line K-562) on the second day of cultivation. At the same
time, the survival rate of cells was virtually unchanged.
Therefore, at the given concentration, it considerably suppressed
the proliferation of cancer cells, without influencing their
survival rate. Baricault L., et al. (1995) reported that probiotics
can initiate the mechanisms of cancer cell differentiation.
Therefore, it is possible that product of the present invention
could switch on mechanisms that control the cell cycle and cell
differentiation. The differentiation of leukemia cells after
treatment with the product is not ruled out. TABLE-US-00001 TABLE 1
Influence of the product of the present invention on survival and
index of proliferation of human chronic myeloid leukemia cells,
line K-562. Time of cultivation Fraction of cells Index of Variant
(days) survived (%) proliferation Control 1 84 .+-. 2 1.19 .+-.
0.06 Product 1 80 .+-. 1 0.79 .+-. 0.09 Control 2 85 .+-. 1 3.35
.+-. 0.15 Product 2 77 .+-. 1 1.71 .+-. 0.07 * * values differs
significantly (p < 0.01) from that observed for appropriate
control.
[0084] Recently, it was demonstrated that the product of the
present invention can act as a neuronal growth factor (it induced
an irreversible differentiation of cancerous PC-12 cells into
neuron-like structures) and manifested clear pharmacological
reactions at the cellular level, i.e. directly activated PC-12
cells and neurons by the release of Ca.sup.2+ from the
intracellular stores in a steady manner and also stimulated the
entry of Ca.sup.2+ into the cells (Sobol et al., (2005), 37:284-293
and J. Neurochem. (2005), 94. (Suppl. 1): 85, 94). Additionally, we
found out that it demonstrated antitumor action against
lymphosarcoma of Pliss (rat), although significant tumor growth
inhibition was observed over only a short period after the start of
the study (unpublished).
Micronucleus Test
[0085] In Toxicological Principles for the Safety of Food
Ingredients (Redbook, 2000, U.S. Food and Drug Administration), it
is recommended to perform a micronucleus test, the purpose of which
is to identify substances that cause cytogenetic damage, resulting
in the formation of micronuclei (MN). Micronuclei arise from two
important types of genetic damage (clastogenesis and spindle
disruption), and the micronucleus assay has been widely used to
screen for chemicals that cause this type of damage. An in vitro
micronucleus assay of the product of the present invention was
performed.
[0086] Methods Blood samples were taken with heparin from 9 healthy
human donors aged between 25 and 45. Lymphocytes were separated on
ficoll-hypaque gradient, washed twice, resuspended in 5 ml RPMI
1640 medium and irradiated (Cs-137) at 2 Gy and 5 Gy at a dose rate
of 1.3 Gy/minute in plastic vessels. Then, irradiated lymphocytes
were resuspended in 5 ml RPMI 1640 medium with Hepes buffer,
containing 30% foetal bovine serum, standard antibiotics,
phytohemagglutinin (PHA, 9 .mu.g/ml) and 0.5 percent of the product
of the present invention added in 60 min after irradiation. The
cells were cultured at 37.degree. C. for 72 hours in closed
vessels. At the end of the cultivation, the cells were collected by
centrifugation, washed, incubated in 0.075 mmol/l of KCl solution
for 7 min at 20.degree. C., fixed in ice-cold 3:1 methanol-acetic
acid, transpired to glass slides and stained with Giemsa.
Cytochalasin B (9 .mu.g/ml) was added 46 hours after PHA
stimulation. The cells were analyzed using an "Axiomat" (Opton,
FRG) microscope at 1000 magnification. Micronuclei (MN) were scored
in 1000 binuclear cells.
[0087] Results: For the nine donors, the micronuclei yield per cell
was 0.212.+-.0.038 at 2 Gy and 0.324.+-.0.027 at 5 Gy for untreated
cells and 0.134.+-.0.034 and 0.162.+-.0.015 for cells treated with
0.5% concentration of the product of the present invention,
respectively. There was a significant difference (p<0.001,
unpaired) between the cells untreated and treated with the product
of the present invention. Thus, in irradiated human lymphocytes,
the product of the present invention reduced MN formation by about
50%, when introduced 1 hour after irradiation. It should be noted
that the total number of MN decreased, mainly because of fewer
cells with more than two MN (known to be a sensitive index of
irradiation). In non-irradiated cells, the product of the present
invention also reduced MN, by about 30 percent.
[0088] Conclusion Clear antimutagenic activity of the product of
the present invention was demonstrated. Therefore, it can be
supported that products of metabolism from friendly bacteria can
reduce mutations occurring in the human genome.
Antimicrobial Activity Assays In-Vitro Against Various
Pathogens.
[0089] Assays were performed to evaluate the antimicrobial activity
of product of present invention. In Table 2, this effect on various
pathogens is presented. TABLE-US-00002 TABLE 2 Some antimicrobial
activities of product medium against bacteria and viruses. Dilution
of Type of a bacteria or virus product (%) Effect observed M. of
tuberculosis 1/10 (10%) * Growth inhibition Staph. Aureus 1/10
(10%) Bactericidal action (including resistance forms (pH
neutralized) Within 3 hours of bacteria, isolated from blood
samples of patient of hospital) Ps. Aeruginose 1/40 (2.5%)
Bactericidal action (pH neutralized) Within 8 hours Viruses of
influenza 1/100 (1%) Antiviral action Within 48 hours * M.
Tuberculosis virtually insensitive to low pH.
[0090] These studies demonstrated that product of present invention
suppresses the growth of various pathogens.
Microbiological Contamination of Product.
[0091] The following microorganisms were tested for possible
contamination in the product of the present invention (see Table
3). TABLE-US-00003 TABLE 3 Microbiological test of product. Russian
Pharmacological Requirements of N Microorganisms Tested Jun. 1,
1996 Results of Test 1 Total aerobic bacteria in 18 .times.
10.sup.4 aerobic one g of PP tested bacteria in one g of PP* 2
Total quantity of yeast Absence And mold present 3 E-coli Absence
Absence 4 Salmonella Absence Absence 5 Other intestinal bacteria No
more than Absence 100 6 St. Aureus Absence Absence 7 Ps. Aeruginosa
Absence Absence 8 Total bacteria and molds *aerobic bacteria are
Lactobacillus bacteria.
[0092] Conclusion: Based on established scientific and technical
criteria, product of present invention conforms to microbiological
requirements as being without any pathogens.
[0093] Thus, the product of this invention has various unique
properties in comparison to probiotic and their fermentative
products now present in the market. No pathogens can survive in the
product of this invention, even in an unsterile environment. The
shelf-life of the product of the present invention is no less than
several weeks at room temperature and no less than two years below
10 Celsius. The product of the present invention has clear
pharmacological activity at the cellular level on various excitable
and non-excitable cells. It may act as a neuronal growth factor and
effectively suppresses a wide spectrum of pathogens, including
drug-resistance forms (Staphylococci and M. tuberculosis). The
product of the present invention may be effectively applied to
various parts of the human body and utilized without the need for
gastrointestinal digestion. The Lactobacillus strains living in the
presented product are highly active and tolerant of acidic
conditions. Application of the product of the present invention was
effective when conventional medical therapy had failed (see cases 1
and 3).
Prophylaxis and Treatment of Vaginal Diseases
[0094] The liquid medium of the present invention is ideally suited
(prophylaxis and treatment) for vaginal diseases and may be useful
against sexually transmitted infections. On the one hand, it has a
wide spectrum of antiviral and antibacterial activity against
pathogens. On the other hand, it does not disturb the natural
lactobacillus microflora (predominant flora in the vagina), because
it is the product of various natural food components fermented by
Lactobacillus. The low pH of the product of the present invention
(around 3.0) is unsuitable for vaginal pathogens. For AIDS and
tuberculosis patients in Russia, intra-vaginal application (2-3
times a week for around two months) was very useful against
opportunistic infections for women undergoing HAART or antibiotic
therapy and ideally supported such therapy. All patients reported
improved well being with therapy. Indications of infections
appearing in the urine disappeared within 2-4 weeks after
intra-vaginal application. Cystitis disappeared within several
days. Moreover, within 2-3 months of such vaginal treatment using
the products of the present invention, some young women (28-40)
restored their menstrual cycles, which had been absent for
approximately one year due to their disease. Further, because the
product of the present invention may cause differentiation of
cancer cells, has anti-tumour properties and stimulates immunity,
it is effective against cancer of the uterus, ovary and
endometriosis. However, application of product of the present
invention for these diseases should be prolonged for some months
and up to one year. Laparoscopy in women with endometriosis showed
considerable abatement of this process (relief of endometriosis
pain, inhibition the growth of the endometrium and to some extent
restoration of normal anatomy)
[0095] The hydrolyzed fermented medium of the invention is
effective in treating a variety of infectious viral diseases
including Hepatitis A, B, and C; myxoviruses and influenza; herpes
of various types; virus of poliomyelitis; adenoviruses; various
types of encephalitis; proteus, and foot-and-mouth disease. The
medium is effective in treating human immunodeficiency virus (HIV)
and AIDS. It can be used for Ebola, smallpox, Congo-Crimean
hemorrhagic fewer, and yellow fewer.
[0096] Bacteria caused infections can also be treated with the
medium of the invention and include tuberculosis; leprosy; cholera;
various forms of meningitis and Legionnaire's disease; syphilis;
gonorrhea; Lyme disease; typhus; various Streptococcuses and
Staphylococcuses; anthrax; botulism; diphtheria; gangrene; tetanus;
tularemia; chamydiae; plague; mycoplasmas; pathogenic E-coli; etc.
The medium is also effective in treating septic shock, toxic shock,
and multiple organ failure.
[0097] The medium of the invention is also effective against
various fungi including Candida, Pneumocystis, and various other
lung infections.
[0098] The product of the invention is effective for Protozoa
including various types of Plasmodium of malaria; Leishmaniasis;
various Trypanosomas; Cryptosporidium, Toxoplasmosis, and Isospora,
as wells as against various parasites of cattle. It can be
successfully used against helminth infections. The medium is useful
in treating cancer including advanced cancer such as bladder
cancer, breast cancer, colon cancer, gastrointestinal cancers, head
and neck cancers, kidney cancer, leukemia, lymphogranulomatosis,
liver cancer, lymphoma, lung cancer, prostate cancer, ovary cancer,
skin cancers, thymus cancer, thyroid cancers, tongue cancer, vagina
cancer and uterus cancer. The medium is further found to be
effective for cardiovascular diseases including various insults and
strokes (including paralysis); myocardial infarction including the
use as a prophylactic agent and after infarction for patient
recovery; cerebral thrombosis; myocarditis and aneurysms; ischemia;
arteriosclerosis; coronary artery disease; hypertension;
rheumatism; various abnormalities of blood coagulation system.
Another area of use in medicine is for kidney and liver diseases
including dialysis; pyelonephritis, kidney colic; stones in kidney
and liver; hemolytic jaundice.
[0099] The medium of the invention is also effective in treating
hemolytic jaundice, various vaginal diseases, endometriosis,
vaginal cancer, various urogenital tract infections, such as
bacterial vaginosis, yeast vaginitis, and treatment to reduce the
risk of HIV infection upon initial exposure.
[0100] Further areas of medical use include treatment of sexual
dysfunctions and sexual diseases (vaginitis, urethritis, bladder
infection, etc.); for relief of side effects of menopause; for
treating endometriosis; psoriasis; bronchitis; all types of pain
including chronic pain; gingivitis and parodontosis; atrophy;
dystrophy; omphalitis, otitis, sinusitis, and rhinitis; and for
reduction in cholesterol level.
[0101] The medium of the invention can be used both alone and in
conjunction with pharmaceutical drugs as adjuvant therapy. The
administration of the medium reduces considerably the toxic side
effects of conventional therapy.
[0102] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of the principles and
applications of the present invention. It is therefore to be
understood that numerous modifications may be made to the
illustrative embodiments and that other arrangements may be devised
without departing from the spirit and scope of the present
invention as defined by the appended claims.
* * * * *