US20080102061A1

US20080102061A1 - Use hydrolyzed medium containing microorganisms medicinally

Info

Publication number: US20080102061A1
Application number: US11/970,060
Authority: US
Inventors: Constantin Sobol; Yuzefa Sobol
Original assignee: Technology Commercialization Corp
Current assignee: Technology Commercialization Corp
Priority date: 2002-06-21
Filing date: 2008-01-07
Publication date: 2008-05-01
Also published as: CA2484639C; US6953574B2; WO2004000038A1; AU2003236536A1; CA2484639A1; US20030235559A1

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.

Description

CROSS-REFERENCE DATA

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

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.
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

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 T⁰, 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° 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.
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”.
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.
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).
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
Probiotic bacteria produce enzymes that:

- 1) break down proteins into amino acids and peptides,
- 2) break down fats and complex sugars, and
- 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, Jiménez-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 T⁰, because such concentration of lactate is better at suppressing pathogens effectively. However, high acidity (above 800 T⁰) may be toxic to some extent.

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.
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

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.
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.
A further object of the present invention is to provide a medium of high acidity, at least above 3% of lactate concentration (>300 T⁰or 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.
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.
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”.
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.
It is a further yet objective to provide a medium capable of producing long-term therapeutic effects on a human being or an animal.
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.
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.
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 10⁵to 10⁶live 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 T⁰, preferably between 500 to 800 T⁰, 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.
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.
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.
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.
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.
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

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 T⁰), low pH (from 1.5 to 6.5), high concentration of the metabolic products and relatively low concentration of microorganisms (from 10⁵to 10⁶cells/ml) as opposed to the preparations described by others.
In the most basic form, the medium of the invention can be prepared following these steps:

1. Provide a food product of plant nature, wash it and cut into small pieces (dimension of pieces can vary from about ½ 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.
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.
3. Mix the food ingredient with the liquid ingredient in proportions of 10-90% liquid to 70-5% food by weight.
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.

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.
The fermentation endpoint is determined by measuring the acidity in Terner degree. A satisfactory acidity for the medium is between 300 and 900 T⁰, and pH ranges between 1.5 to 6.5.
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 T⁰.
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:

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.
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 10⁷to 10⁹per ml of liquid, and pH is maintained close to neutral. Optionally, fermentation in water or juice can also be used.

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.
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.
Preferred strains of Bifidobacteria include Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium cereus, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, and Bifidobacterium thermophilus.
Streptococci strains to be used include preferably Streptococcus lactis, Streptococcus cremoris, Streptococcus diacetylactis, Streptococcus thermophilus, and Streptococcus faecium.
Preferred yeast includes Saccharomyces boulardii, Saccharomyces cerevisiae, and Saccharomyces lactis.
Propionic bacteria strain is preferably propionibacterium shermanii.
Pedicocci strains that may be used in the present invention include Pediococcus cerevisia, Pediococcus acidilactici and Pediococcus pentosaceus.
Leuconostoc strains include Leuconostoc cremoris, Leuconostoc dextranicum, and Leuconostoc mesenteriodes.

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.

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.

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).
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.
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.
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.
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 10⁵to 10⁶per 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.

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.
The acidity of the final liquid medium product is 300 to 900 T⁰, preferably between 500 to 800 T⁰, 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.
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.
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.
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.
The following are examples of specific use of the medium of the invention. All patients were diagnosed and treated in Russian hospitals.
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.
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.
MEDICAL PROGNOSIS: not favorable.
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.
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.
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.
MEDICAL PROGNOSIS: not favorable.
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.
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.
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^thday after surgery, the swallowing reflex appeared. By the 50^thday, the patient gained in weight by 2.5 kg. On 53^rdday, 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
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 NaHCO₃. 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.

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 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.

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 Ca²⁺ from the intracellular stores in a steady manner and also stimulated the entry of Ca²⁺ 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
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.
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 μg/ml) and 0.5 percent of the product of the present invention added in 60 min after irradiation. The cells were cultured at 37° 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° C., fixed in ice-cold 3:1 methanol-acetic acid, transpired to glass slides and stained with Giemsa. Cytochalasin B (9 μg/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.
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.
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.

Assays were performed to evaluate the antimicrobial activity of product of present invention. In Table 2, this effect on various pathogens is presented.

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.

These studies demonstrated that product of present invention suppresses the growth of various pathogens.
Microbiological Contamination of Product.

The following microorganisms were tested for possible contamination in the product of the present invention (see Table 3).

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 × 10⁴aerobic
	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.

Conclusion: Based on established scientific and technical criteria, product of present invention conforms to microbiological requirements as being without any pathogens.
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
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)
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.
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.
The medium of the invention is also effective against various fungi including Candida, Pneumocystis, and various other lung infections.
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.
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.
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.
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.
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.

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 T⁰, 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 T⁰;

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 T⁰.

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.