WO1999007393A1 - Pharmaceutical product comprising bacterial strains for the colon therapy - Google Patents

Pharmaceutical product comprising bacterial strains for the colon therapy Download PDF

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Publication number
WO1999007393A1
WO1999007393A1 PCT/RO1998/000015 RO9800015W WO9907393A1 WO 1999007393 A1 WO1999007393 A1 WO 1999007393A1 RO 9800015 W RO9800015 W RO 9800015W WO 9907393 A1 WO9907393 A1 WO 9907393A1
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Prior art keywords
parts
strain
lactobacillus
ferments
vitamin
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PCT/RO1998/000015
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French (fr)
Inventor
Rodica Teodorescu
Original Assignee
Rodica Teodorescu
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Publication date
Application filed by Rodica Teodorescu filed Critical Rodica Teodorescu
Priority to AU87540/98A priority Critical patent/AU8754098A/en
Publication of WO1999007393A1 publication Critical patent/WO1999007393A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis

Definitions

  • the present invention relates to a medicinal product for the colon therapy, having special effects in the postoperative treatment of colon cancer.
  • the active element in this action is the lactic acid and other specific substances with antibiotic action of nisine type.
  • the nisine is characterized by a strong inhibitory action of species belonging to the putrefaction microflora (Aerobacter sp., Esche ⁇ chia sp., Proteus sp., Stafilococcus sp.) either by blocking the breathing and their metabolism or by stopping the cell division.
  • lactobacilli have been used in intestinal diseases therapy, as preparations comprising associated or non-associated monomicrobiai cultures with other medicinal substances.
  • the eubiotics are pure cultures of lactic bacteria, especilly selected Lactobacillus acidophylus strains that normally live in the digestive tube and Lactobacillus casei found in the saliva.
  • compositions comprising different Lactobacillus strains used in human medicine for the treatment of different diseases.
  • the researches for obtaining eubiotics of great efficiency were intended to obtain new products, having a greater acid power that ensures the destruction of putrefaction flora as well as the realization of optimum conditions for the growth of normal saprophytic flora. It was, also, intended to obtain, by selection, some Lactobacillus strains that release, in the medium, increased amounts of antibiotic substances such as the lactobaciline, lactocitine as well as ergons, having good effects on the living cell, stimulating themputation of several endogenous systems and increasing the vital capacity.
  • the medicinal product is constituted of 0,093650 parts mixture of 4 strains: Lactobacillus acidophylus-LAR, Lactobacillus casei-LC, Lactobacillus piantarum-LP ⁇ and Streptococcus lactis-SLR registered under the nos. 31 , 27, 10 and 36 in the Biosan Centre Collection (0,5 : 0.5 : 2,5 : 0,5), 40 parts vitamin B,, 10 parts of vitamin B 2 , 100 parts of vitamin B 6 200 parts of calcium panthotenate and 6000 parts L-lysine hydrochlorate, the parts being expressed in weight.
  • the Lactobacillus acidophylus-LAR no.31 strain is a non-spinning strain, with an acid power of 148 ⁇ and ferments the raff ⁇ nose and trehalose with a maximum efficiency.
  • the Lactobacillus casei-LC no.27 strain has an acid power of 185 °T and ferments the rhamnose.
  • the Lactobacillus plantarum-LP ⁇ no.10 strain coagulates the milk without eliminating the whey, forms a rich deposit on a synthetic medium, has an acid power of 150°T and ferments the xylose.
  • the Streptococcus lactis-SLR no.36 strain ferments the esculin.
  • the following components are associated: 93,650 g of lyophilised powder obtained by mixture of the 4 bacterial strains (association ratio 0,5 : 0,5 : 2,5: 0,5) is mixed, at cold, with 40 mg of vitamin B 1; 10 mg of vitamin 2 B , 100 g of vitarrjin B , 200 mg of calcium panthotenate and 6000 mg of L-lysine hydrochlorate.
  • the pure Lactobacillus acidophylus strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the lactobacilli strain was introduced in liquid MRS medium and was thermostated at 37 ⁇ £ during 24...48 hours.
  • 1% 0 peptoned water was introduced in 10 sterile test glasses; in the first test glass, 10 ml and 9 mi in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining .isolated colonies, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised MRS medium at 45% were poured. In was homogenized very well to obtain uniformly dispersed colonies.
  • the Petri plate was introduced in thermostate at 371, during 48 hours, the lid downwards. With a handle without ringlet, the colonies that we were interested in were isolated, taking into consideration that the aspect of colonies is specific to the Lactobacillus acidophylus species.
  • the pure Lactobacillus casei strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the lactobacilli strain was introduced in liquid MRS medium and was thermostated at 37°C during 24...48 hours.
  • 1 % 0 peptoned water was introduced in 10 sterile test glasses; in the first test glass, 10 ml and 9 ml in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining isolated strains, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised MRS medium at 45 °C were poured. In was homogenized very well to obtain uniformly dispersed colonies. After the medium became solid, the Petri plate was introduced in thermostate at 37 ⁇ , during 48 hours, the lid downwards.
  • the pure Lactobacillus plantarum strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the lactobacilli strain was introduced in liquid MRS medium and was thermostated at 37°C during 24...48 hours.
  • 1% 0 peptoned water was introduced in 10 test glasses; in the first test glass, 10 ml and 9 ml in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining isolated colonies, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised MRS medium at 45 O were poured. In was homogenized very well to obtain uniformly dispersed colonies.
  • the Petri plate was introduced in thermostate, during 48 hours, the lid downwards. With a handle without ringlet, the colonies that we were interseted in were isolated, taking into consideration that the aspect of colonies is specific to the Lactobacillus plantarum species.
  • the pure Streptococcus lactis strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the Streptococcus lactis strain was introduced in hydrolized milk with extract of yeast (LHD) and was thermostated at 24°C during 24...48 hours.
  • 1 % 0 peptoned water was introduced in 10 sterile test glasses; in the first test glass, 10 ml and 9 ml in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining isolated colonies, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised LHD medium at 45 ⁇ were poured. In was homogenized very well to obtain uniformly dispersed colonies.
  • the Petri plate was introduced in thermostate at 24 O, during 48 hours, the lid downwards. With a handle without ringlet, the colonies that we were intersted in were isolated, taking into consideration that the aspect of colonies is specific to the Streptococcus lactis species.
  • Lactobacillus casei strain used in product, according to the invention, in comparison with those of Lactobacillus casei common strains.
  • Streptococcus lactis strain used in product, according to the invention in comparison with those of Streptococcus lactis common strains.
  • lactobacillus and streptococcus strains according to the invention, to different concentrations of antibiotics and chemi-therapeutics.
  • strains from the drug composition, according to the invention were adapted to the following antibiotics:
  • the antibioresistance is verified by antibiogramme on solid base medium by the usual microtablets.
  • the medium is inseminated by incorporation, it is let to become solid, then, the microtablets are placed at 37°C, 48...72 hours.
  • the isolated strains are maintained on synthetic media such as MRS and LHD whose composition is known per se.
  • the strains Periodically, the strains are submitted to the most important tests known for identification with a view to establishing the resistance in time of characteristics as well as their ageing speed.
  • the strains are periodically passed on known solid media.
  • the sterile degreased milk is also the protecting medium used for the last stage of the process, namely the lyophilisation.
  • a pure polymicrobial culture will be obtained.
  • the polymicrobial culture obtained according to the invention, is coagulated at - 45°C - 50°C, then the proper lyophilsation starts in vacuum of 3 torrs, during 30 hours, having the sterile degreased milk as protection medium, according . to the standard requirements for alimentary milk.
  • the polymicrobial lyophilised powder has the following characteristics:
  • pancreatitis
  • the product is administered per os 30 minutes before the three main meals, during 7...20 days, depending on the disease gravity.
  • the product can be administered alone or associated with antibiotics ⁇ chemico-therapeutics or intestinal dressings.
  • the dose is of 50...100 mg of product according to the invention.
  • the action of the medicinal product appears at the moment of colics disappearance, of consistency normalization and of stools aspect as well as at a better tolerance at some foods that, before treatment, were not tolerated.
  • the eubiotic effect has been had in view in diseases of inflammatory and degenerative type.

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  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
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  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

The medicinal product, according to the invention, is constituted of 0,093650 parts mixture of 4 strains: Lactobacillus acidophylus - LAR, Lactobacillus casei - LC, Lactobacillus plantarum - LPI and Streptococcus lactis - SLR, registered under numbers 31, 27, 10 and 36 in the Biosan Centre Collection (0,5 : 0,5 : 2,5 : 0,5), 40 parts of vitamin B1, 10 parts of vitamin B2, 100 parts of vitamin B6, 200 parts of calcium panthotenate and 6000 parts of L-lysine hydrochloride, the parts being expressed in weight. The Lactobacillus acidophylus - LAR no.31 strain is a non-spinning strain, with an acid power of 148 °T and ferments, at maximum efficiency, the raffinose and trehalose. The Lactobacillus casei - LC no.27 strain has an acid power of 185 °T and ferments the rhamnose. The Lactobacillus plantarum - LPI no.10 strain coagulates the milk without eliminating the whey, forms a rich deposit on a synthetic medium, has an acid power of 150 °T, ferments the xylose. The Streptococcus lactis - SLR no.36 strain ferments the esculin.

Description

PHARMACEUTICAL PRODUCT COMPRISING BACTERIAL STRAINS FOR TOE COLON THERAPY
The present invention relates to a medicinal product for the colon therapy, having special effects in the postoperative treatment of colon cancer.
At the same time with the introduction of antibiotics and chemi-therapeutics, very good results have been obtained as regards the pathogenic agent of different diseases. Nevertheless, the consequences, in cases of prolonged administration or repeated use of antibiotics, have been the most unpleasant ones.
It is known that, during the oral use of antibiotics, in digestive disorders, with specified or unspecified germs, there appear resistance phenomena of these ones. Therefore, the effect of the antibiotic diminishes, the normal intestinal flora is destroyed and the putrefaction flora as well as the pathogenic germs further grow.
The researches for finding natural remedies have been extensively developed worldwide. The institutions of high reputation such as the "Experimental Centre of Milk" and the "Country Institute for Infantile Protection" of Milano have published studies on the variation of lactic flora and intestinal bifides. These studies have shown that lactobacilli from yoghurt survive to the passage by the digestive tract, stopping the growth of putrefaction flora and helping the multiplication of autochthonous lactic and bifide flora.
The problem regarding the use of some lactobacilli species for destroying the putrefaction flora was raised many years ago. The active element in this action is the lactic acid and other specific substances with antibiotic action of nisine type. The nisine is characterized by a strong inhibitory action of species belonging to the putrefaction microflora (Aerobacter sp., Escheήchia sp., Proteus sp., Stafilococcus sp.) either by blocking the breathing and their metabolism or by stopping the cell division.
Therefore, the lactobacilli have been used in intestinal diseases therapy, as preparations comprising associated or non-associated monomicrobiai cultures with other medicinal substances.
The researches performed in the "Biosan" Centre have made as results the obtaining of biolgical products based on different combinations of lactobacilli specific to the digestive tract.
The clinical tests and, afterwards, the clinical use of "Biosan" Centre products have shown that eubiotics are based on strains of great enzymatic vitality and power, are considered as a therapeutical way of inestimable value representing a therapy of intestinal microbiological substitution of great efficiency. It results the necessity of precise rules for the production and,control of these preparations.
The eubiotics are pure cultures of lactic bacteria, especilly selected Lactobacillus acidophylus strains that normally live in the digestive tube and Lactobacillus casei found in the saliva.
During the researches performed at the "Biosan" Centre, three generations of eubiotics were prepared and, afterwards, manufactured, medicinal products where selected Lactobacillus acidophylus, Lactobacillus casei and Lactobacillus plantarum strains were associated.
There are known pharmaceutical compositions comprising different Lactobacillus strains used in human medicine for the treatment of different diseases.
The researches for obtaining eubiotics of great efficiency were intended to obtain new products, having a greater acid power that ensures the destruction of putrefaction flora as well as the realization of optimum conditions for the growth of normal saprophytic flora. It was, also, intended to obtain, by selection, some Lactobacillus strains that release, in the medium, increased amounts of antibiotic substances such as the lactobaciline, lactocitine as well as ergons, having good effects on the living cell, stimulating the fonction of several endogenous systems and increasing the vital capacity.
The medicinal product, according to the invention, is constituted of 0,093650 parts mixture of 4 strains: Lactobacillus acidophylus-LAR, Lactobacillus casei-LC, Lactobacillus piantarum-LP\ and Streptococcus lactis-SLR registered under the nos. 31 , 27, 10 and 36 in the Biosan Centre Collection (0,5 : 0.5 : 2,5 : 0,5), 40 parts vitamin B,, 10 parts of vitamin B2, 100 parts of vitamin B6 200 parts of calcium panthotenate and 6000 parts L-lysine hydrochlorate, the parts being expressed in weight.
The Lactobacillus acidophylus-LAR no.31 strain is a non-spinning strain, with an acid power of 148^ and ferments the raffϊnose and trehalose with a maximum efficiency.
The Lactobacillus casei-LC no.27 strain has an acid power of 185 °T and ferments the rhamnose.
The Lactobacillus plantarum-LP\ no.10 strain coagulates the milk without eliminating the whey, forms a rich deposit on a synthetic medium, has an acid power of 150°T and ferments the xylose.
The Streptococcus lactis-SLR no.36 strain ferments the esculin.
The advantages of the medicinal product, according to the invention, are the following:
- it acidifies the medium with prolonged effect; - it inhibits the effect of hyaluronidase stopping the spreading of pathogenic bacteria, influencing the permeability of intestinal mucous membrane;
-it reduces the activity of gluconronidase, azoreductase and nitrureductase, substances having an homogenous action on the cell;
- it stimulates the non-oncogenical cell mitosis, with a view to regenerating the intestinal mucous membrane.
It is given below an example of realization of the invention.
For obtaining 100 g of medicinal product, according to the present invention, the following components are associated: 93,650 g of lyophilised powder obtained by mixture of the 4 bacterial strains (association ratio 0,5 : 0,5 : 2,5: 0,5) is mixed, at cold, with 40 mg of vitamin B1; 10 mg of vitamin2B , 100 g of vitarrjin B , 200 mg of calcium panthotenate and 6000 mg of L-lysine hydrochlorate.
The addition and mixture of ingredients is performed in conditions of protection against hygroscopicity and the packing is realized in dark coloured containers, with drier powders in the closing system that should be tight.
Modality for obtaining the strains a) Lactobacillus acidophylus
The pure Lactobacillus acidophylus strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the lactobacilli strain was introduced in liquid MRS medium and was thermostated at 37 <£ during 24...48 hours.
Working modality
1%0 peptoned water was introduced in 10 sterile test glasses; in the first test glass, 10 ml and 9 mi in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining .isolated colonies, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised MRS medium at 45% were poured. In was homogenized very well to obtain uniformly dispersed colonies. After the medium became solid, the Petri plate was introduced in thermostate at 371, during 48 hours, the lid downwards. With a handle without ringlet, the colonies that we were interested in were isolated, taking into consideration that the aspect of colonies is specific to the Lactobacillus acidophylus species. The isolated strains were introduced in the test glasses with a liquid MRS medium having the following composition: 10°/ 00 of tryptone, 10°/00 of extract of meat, 10°/00 of extract of yeast -pH = 6...6,5, 20°/00 of glucose, 1 °/00 of Tween 80, 2°/00 of dipotassic phosphate, 5°/00 of sodium acetate, 2°/00 of ammonium citrate, 0,2°/00 of magnesium sulphate, 0,05°/00 of manganese sulphate. It is sterilized 20 minutes at 1 atmosphere. b) Lactobacillus casei
The pure Lactobacillus casei strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the lactobacilli strain was introduced in liquid MRS medium and was thermostated at 37°C during 24...48 hours.
Working modality
1 %0 peptoned water was introduced in 10 sterile test glasses; in the first test glass, 10 ml and 9 ml in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining isolated strains, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised MRS medium at 45 °C were poured. In was homogenized very well to obtain uniformly dispersed colonies. After the medium became solid, the Petri plate was introduced in thermostate at 37^, during 48 hours, the lid downwards.
With a handle without ringlet, the colonies that we were interested in were isolated, taking into consideration that the aspect of colonies is specific to the Lactobacillus casei species.
The isolated strains were introduced in test glasses with liquid MRS medium (Man, Rogoza, Sharpe medium) having the known composition. c) Lactobacillus plantarum
The pure Lactobacillus plantarum strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the lactobacilli strain was introduced in liquid MRS medium and was thermostated at 37°C during 24...48 hours.
Working modality
1%0 peptoned water was introduced in 10 test glasses; in the first test glass, 10 ml and 9 ml in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining isolated colonies, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised MRS medium at 45 O were poured. In was homogenized very well to obtain uniformly dispersed colonies. After the medium became solid, the Petri plate was introduced in thermostate, during 48 hours, the lid downwards. With a handle without ringlet, the colonies that we were interseted in were isolated, taking into consideration that the aspect of colonies is specific to the Lactobacillus plantarum species.
The isolated strains were introduced in test glasses with liquid MRS medium having the known composition. d) Streptococcus lactis
The pure Streptococcus lactis strain was obtained by repeated dispersions and isolations performed as follows: the sample from which we want to isolate the Streptococcus lactis strain was introduced in hydrolized milk with extract of yeast (LHD) and was thermostated at 24°C during 24...48 hours.
Working modality
1 %0 peptoned water was introduced in 10 sterile test glasses; in the first test glass, 10 ml and 9 ml in other ones. From the previously impregnated medium, it is taken with a handle, introducing it in the first test glass. It was homogenized very well with a dropper of 1 ml and successive dilutions were performed following the known technique. For obtaining isolated colonies, it was gone as far as to obtain the dilution 6 - 7, from which 1 ml was placed on a Petri plate, over which 15...20 ml of agarised LHD medium at 45 ^ were poured. In was homogenized very well to obtain uniformly dispersed colonies. After the medium became solid, the Petri plate was introduced in thermostate at 24 O, during 48 hours, the lid downwards. With a handle without ringlet, the colonies that we were intersted in were isolated, taking into consideration that the aspect of colonies is specific to the Streptococcus lactis species.
The isolated strains were introduced in test glasses with liquid LHD medium having the following composition: 100 ml of concentrated hydrolized milk pH=6,5...6,8, 100 ml of distilled water, 5 g of autholysis of yeast, 1 g of sodium acetate. It is sterilized 15 minutes at 120°C.
It is shown below the characteristics of isolated and identified strains in comparison with the common strains of the respective species. The characteristics of Lactobacillus acidophylus strain used in product, according to the invention, in comparison with those of Lactobacillus acidophylus common strains.
Figure imgf000008_0001
Figure imgf000009_0001
Figure imgf000010_0001
The characteristics of Lactobacillus casei strain used in product, according to the invention, in comparison with those of Lactobacillus casei common strains.
Figure imgf000010_0002
Figure imgf000011_0001
Figure imgf000012_0001
Figure imgf000013_0001
The characteristics oi Lactobacillus plantarum strain used in product, according to the invention, in comparison with those of Lactobacillus plantarum common strains.
Figure imgf000013_0002
Figure imgf000014_0001
Figure imgf000015_0001
The characteristics of Streptococcus lactis strain used in product, according to the invention, in comparison with those of Streptococcus lactis common strains.
Figure imgf000016_0001
Figure imgf000017_0001
Figure imgf000018_0001
The adaptation of lactobacillus and streptococcus strains, according to the invention, to different concentrations of antibiotics and chemi-therapeutics.
The strains from the drug composition, according to the invention, were adapted to the following antibiotics:
Figure imgf000018_0002
Alternative passages were performed on liquid MRS medium with and without antibiotics, in progressive amounts.
An example of adaptation for an antibitiotic - penicillin is given below.
A solution of penicillin with 100 Ul/cc is prepared being added to the sterile base medium, in the following amounts: passage I 0,5 Ul/cc VIII 4 Ul/cc
II 1 Ul/cc IX 4,5 Ul/cc
III 1 ,5 Ul/cc" X 5 Ul/cc IV 2 . XI 5,5 Ul/cc
V 2,5 Ul/cc XII 6 Ul/cc
VI 3 Ul/cc XIII 6,5 Ul/cc
VII 3,5 UI/cc XIV 7 Ul/cc
For maintaining the resistance at antibiotics, passages are further performed on the base medium, with the maximum dose of antibiotics (7 Ul/cc).
The same procedure is for all antibiotics.
The antibioresistance is verified by antibiogramme on solid base medium by the usual microtablets. The medium is inseminated by incorporation, it is let to become solid, then, the microtablets are placed at 37°C, 48...72 hours.
If clear zones do not appear around the microtablets, the strains are resistant.
The isolated, identified and antibioresistant strains were registered in the Biosan Centre Collection under the numbers: a) Lactobacillus acidophylus - strain LAR no.31 b) Lactobacillus casei - strain LC no.27 c) Lactobacillus plantarum - strain LPI no.10 d) Streptococcus lactis - strain SLR no.36
The isolated strains are maintained on synthetic media such as MRS and LHD whose composition is known per se.
Periodically, the strains are submitted to the most important tests known for identification with a view to establishing the resistance in time of characteristics as well as their ageing speed.
For revitality, the strains are periodically passed on known solid media.
In order to prepare the strains for the production stage, these ones are multiplied, then passed on sterile degreased milk, in their association ratio from the product, according to the invention.
The sterile degreased milk is also the protecting medium used for the last stage of the process, namely the lyophilisation. A pure polymicrobial culture will be obtained.
There have not been noticed antagonism phenomena between the lactobacilli strains, the number of viable germs from the polymicrobial culture being the optimum one for the product according to the invention.
The polymicrobial culture, obtained according to the invention, is coagulated at - 45°C - 50°C, then the proper lyophilsation starts in vacuum of 3 torrs, during 30 hours, having the sterile degreased milk as protection medium, according. to the standard requirements for alimentary milk. The polymicrobial lyophilised powder has the following characteristics:
- aspect - lyophilised powder, without mechanical impurities, hygroscopic;
- colour - white yellowish;
- smell - specific;
- taste - sourish specific - acid;
- solubility - insoluble in water, but it easily disperses, forming a homogenous dispersion;
- pH of suspension 1% in water 3, 8...5
- content in chlorine %/g/g max. 1 ,2
- content in calcium %/g/g min. 0,8
- content in nitrogen %/g/g min. 4,5
- residues by calcination max. 1 ,2
- heavy metals Absent
- number of viable germs min. 4 x 107 germs/0,05 g.
The therapeutical indications of the product, according to the invention:
- severe radicle rectite;
- rectorrhagia and mucous eliminations;
- control of intestinal transit, of pains with rectal tenesmus and of constipations with paintful phenomena;
- ulcerous - haemorrhageous recto-colitis;
- the Crohn disease localized in colon;
- hypomotility;
- severe constipation;
- colon diverticulosis;
- benign and malignant stenoses;
- acute, necrotic, operated pancreatitis;
- biliary lithiasis with septic angiocolitis;
- peritonitis with several etiologies;
- entero-colitis with disbacteria;
- mal-absorptions;
- recovery of normal saprophytic microbial flora. Administration of drug according to the invention
The product is administered per os 30 minutes before the three main meals, during 7...20 days, depending on the disease gravity. The product can be administered alone or associated with antibiotics^ chemico-therapeutics or intestinal dressings. The dose is of 50...100 mg of product according to the invention.
The action of the medicinal product appears at the moment of colics disappearance, of consistency normalization and of stools aspect as well as at a better tolerance at some foods that, before treatment, were not tolerated.
The eubiotic effect has been had in view in diseases of inflammatory and degenerative type.
Figure imgf000021_0001
The diagnosis of diseases has been determined following a:
- clinical examination 30%
- bio-chemical examination 70%
- bacteriological examination 80%
- immunological examination 100%
- radiological examination 70%
- irrigoscopical examination 100% Way of administration
Figure imgf000021_0002
Tolerance to drug
Figure imgf000022_0001
Therapeutical charts
Figure imgf000022_0002
The very good evolution after the treatment applied on disease type in comparison with the initial parameters
Figure imgf000022_0003
During the clinical tests, the following benefic effects of the product have been ascertained, according to the invention:
- epithalization of ulcerous lesions in 72% of cases;
- the rapid solution of some postoperative disbacteriosis or of some colopaties after a prolonged treatment with antibiotics;
- a visible improvement of appetite shown at 100% of cases, with the amelioration of general condition of patients and the weight increase;
- elimination of superinfection zones by the closing of cryptic abscesses;
- amelioration of transit in all cases;
- changement of bacterial flora profile;
- it has been also noticed a very good tolerance to the drug.

Claims

C L A I M S
1. A medicinal product for the colon therapy comprising Lactobacillus characterized in that is constituted of 0,093650 parts mixture of 4 strains: Lactobacillus acidophylus - LAR, Lactobacillus casei - LC, Lactobacillus plantarum - LPI and Streptococcus lactis - SLR, registered under numbers 31 , 27, 10 and 36 in the Biosan Centre Collection (0,5 : 0,5 : 2,5 : 0,5), 40 parts of vitamin B ., 10 parts of vitamin B 2 100 parts of vitamin B6, 200 parts of calcium panthotenate and 6000 parts of L-lysine hydrochloride, the parts being expressed in weight.
2. A medicinal product, according to claim 1 , characterized in that the Lactobacillus acidophylus - LAR no.31 strain is a non-spinning strain, with an acid power of 148 ' and ferments, at maximum efficiency, the raffinose and trehalose.
3. A medicinal product, according to claim 1 , characterized in that the Lactobacillus casei - LC no.27 strain has an acid power of 185┬░T and ferments the rhamnose.
4. A medicinal product, according to claim 1 , characterized in that the Lactobacillus plantarum - LPI no.10 strain coagulates the milk without eliminating the whey, forms a rich deposit on a synthetic medium, has an acid power of 150┬░T, ferments the xylose.
5. A medicinal product, according to claim 1 , characterized in that the Streptococcus lactis - SLR no.36 strain ferments the esculin.
PCT/RO1998/000015 1997-08-05 1998-08-03 Pharmaceutical product comprising bacterial strains for the colon therapy WO1999007393A1 (en)

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RO97-01479A RO112995B1 (en) 1997-08-05 1997-08-05 Medicinal product for colon therapy
RO97-01479 1997-08-05

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WO2001070246A1 (en) * 2000-03-24 2001-09-27 Societe Des Produits Nestle S.A. Use of lactic acid bacterium for the treatment of peritonitis
WO2001097821A1 (en) * 2000-06-19 2001-12-27 Mucoprotec Pty Ltd Immunotherapy or treating bacterial or viral infection at mucosal surfaces with probiotics, and compositions therefor.
EP1992351A1 (en) * 2007-05-18 2008-11-19 Nestec S.A. Probiotics in a pre- and/or post-surgical environment
EP2349295A1 (en) * 2008-11-17 2011-08-03 Essum Ab Pharmaceutical preparation comprising a combination of streptococcus strains and lactobacillus strains

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US7678370B2 (en) 2000-03-24 2010-03-16 Nestec S.A. Methods of preventing peritonitis by administering lactic acid bacterium
JP2003527432A (en) * 2000-03-24 2003-09-16 ソシエテ デ プロデユイ ネツスル ソシエテ アノニム Use of lactic acid bacteria for the treatment of peritonitis
AU783439B2 (en) * 2000-03-24 2005-10-27 Societe Des Produits Nestle S.A. Use of lactic acid bacterium for the prevention of peritonitis
AU783439C (en) * 2000-03-24 2006-12-14 Societe Des Produits Nestle S.A. Use of lactic acid bacterium for the prevention of peritonitis
US7217414B2 (en) 2000-03-24 2007-05-15 Nestec S.A. Methods of preventing peritonitis by administering lactic acid bacterium
WO2001070246A1 (en) * 2000-03-24 2001-09-27 Societe Des Produits Nestle S.A. Use of lactic acid bacterium for the treatment of peritonitis
WO2001097821A1 (en) * 2000-06-19 2001-12-27 Mucoprotec Pty Ltd Immunotherapy or treating bacterial or viral infection at mucosal surfaces with probiotics, and compositions therefor.
CN103623010A (en) * 2007-05-18 2014-03-12 雀巢产品技术援助有限公司 Probiotics in a pre- and/or post-surgical environment
WO2008141989A1 (en) * 2007-05-18 2008-11-27 Nestec S.A. Probiotics in a pre- and/or post surgical environment
JP2010527343A (en) * 2007-05-18 2010-08-12 ネステク ソシエテ アノニム Probiotics in pre-operative and / or post-operative environments
RU2470652C2 (en) * 2007-05-18 2012-12-27 Нестек С.А. Probiotic in pre- and/or postoperative period
US8529887B2 (en) 2007-05-18 2013-09-10 Nestec S.A. Probiotics in a pre- and/or post surgical environment
JP2013216658A (en) * 2007-05-18 2013-10-24 Nestec Sa Probiotics in pre- and/or post-surgical environment
EP1992351A1 (en) * 2007-05-18 2008-11-19 Nestec S.A. Probiotics in a pre- and/or post-surgical environment
AU2008253040B2 (en) * 2007-05-18 2014-06-12 Société des Produits Nestlé S.A. Probiotics in a pre- and/or post surgical environment
US9616094B2 (en) 2007-05-18 2017-04-11 Nestec S.A. Probiotics in a pre- and/or post-surgical environment
EP2349295A1 (en) * 2008-11-17 2011-08-03 Essum Ab Pharmaceutical preparation comprising a combination of streptococcus strains and lactobacillus strains
EP2349295A4 (en) * 2008-11-17 2012-12-12 Essum Ab Pharmaceutical preparation comprising a combination of streptococcus strains and lactobacillus strains
AU2009314666B2 (en) * 2008-11-17 2015-01-29 Winclove Holding B.V. Pharmaceutical preparation comprising a combination of Streptococcus strains and Lactobacillus strains
US10596244B2 (en) 2008-11-17 2020-03-24 Winclove Holding B.V. Pharmaceutical preparation comprising a combination of Streptococcus strains and lactobacillus strains

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